Uses of monoclonal antibody 8h9

ABSTRACT

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

This application is a continuation-in-part application of U.S. Ser. No.10/097,558, filed on 8 Mar. 2002; U.S. Ser. No. 10/273,762, filed on 17Oct. 2002; and International Application No. PCT/US02/33331, filed on 17Oct. 2002, the contents of which are incorporated by reference hereintothis application.

The invention disclosed herein was made with government support underDepartment of Energy Grant No. DE-FG-02-93ER61658 (1997-2002), theNational Cancer Institute Grant No. NCI CA 89936 (Dec. 1, 2000-Nov. 30,2002), and National Institutes of Health Grant No. CA61017. Accordingly,the U.S. Government has certain rights in this invention.

Throughout this application, various references are cited. Disclosuresof these publications in their entireties are hereby incorporated byreference into this application to more fully described the state of theart to which this invention pertains.

BACKGROUND OF THE INVENTION

Tumor-restricted surface antigens may be targets for diagnosis andimmune-based therapies. Monoclonal antibody 8H9 is a murine IgG1hybridoma derived from the fusion of mouse myeloma SP2/0 cells andsplenic lymphocytes from BALB/c mice immunized with human neuroblastoma.By immunohistochemistry, 8H9 was highly reactive with human braintumors, childhood sarcomas, neuroblastomas and less so withadenocarcinomas. Among primary brain tumors, 15/17 glioblastomas, 3/4mixed gliomas, 4/11 oligodendrogliomas, 6/8 astrocytomas, 2/2meningiomas, 3/3 schwannomas, 2/2 medulloblastomas, 1/1 neurofibroma,1/2 neuronoglial tumors, 2/3 ependymomas and 1/1 pineoblastoma weretested positive. Among sarcomas, 21/21 Ewing's/PNET, 28/29rhabdomyosarcoma, 28/29 osteosarcomas, 35/37 desmoplastic small roundcell tumors, 2/3 synovial sarcomas, 4/4 leiomyosarcomas, 1/1 malignantfibrous histiocytoma and 2/2 undifferentiated sarcomas tested positivewith 8H9. 87/90 neuroblastomas, 12/16 melanomas, 3/4 hepatoblastomas,7/8 Wilm's tumors, 3/3 rhabdoid tumors and 12/27 adenocarcinomas alsotested positive. In contrast 8H9 was nonreactive with normal humantissues including bone marrow, colon, stomach, heart, lung, muscle,thyroid, testes, pancreas, and human brain (frontal lobe, cerebellum,pons and spinal cord). Reactivity with normal cynomolgus monkey tissuewas similarly restricted. Indirect immunofluorescence localized theantigen recognized by 8H9 to the cell membrane. The antigen isproteinase-sensitive and is not easily modulated off cell surface. 8H9immuno-precipitated a 58 kD band following N-glycanase treatment, mostlikely a protein with heterogeneous degree of glycosylation. This novelantibody-antigen system may have potential for tumor targeting.

Monoclonal antibodies such as 3F8 (1) and 14.18 (2) against G_(D2) inneuroblastoma, M195 against CD33 in acute leukemia (3), anti-HER2antibodies in breast cancer (4) and anti-CD20 antibodies in lymphoma (5)have shown efficacy in recent clinical trials. The prognosis in glialbrain tumors and metastatic mesenchymal and neuroectodermal tumorsremains dismal despite innovations in chemotherapy and radiationtherapy. Immunotherapy may offer new possibilities for improving theoutcome in these patients.

Tumor antigens expressed on cell membrane are potential targets inimmunotherapy. Examples of tumor antigens expressed on glial tumorsinclude neural cell adhesion molecules (6), gangliosides such as G_(D2)and G_(M2) (7), and neurohematopoeitic antigens (8). Recentinvestigations have focused on growth factor receptors as immunetargets, in particular type III mutant epidermal growth factor receptor(EGFRvIII) which has been shown to be expressed on 50% of glial braintumors (9). Notwithstanding the universal expression of NCAM by neuronalcells, two clinical studies have utilized anti-NCAM antibodies inpatients. MAb UJ13A was shown to accumulate in gliomas by virtue ofdisruption of blood brain barrier locally (10) and another antibody,ERIC-1 was used in a therapeutic setting in resected glioma cavitieswith some clinical benefit (11)

Recent studies have targeted immunotherapy to extracellular matrixaround tumor cells. Tenascin has been reported to be expressed in 50-95%of glial brain tumors as well as on mesenchymal tumors, carcinomas andnormal human glial, liver and kidney cells (12). Anti-tenascinmonoclonal antibodies 81C6 (13) and BC-2 and BC-4 (14) administeredintra-cavity have recently been reported to show efficacy in thetreatment of patients with malignant gliomas. However, since theseantigens are also present to varying degrees on normal human neural andnon-neural cells, their clinical utility would depend on theiroverexpression by brain tumors when compared to normal tissues. With theexception of EGFRvIII, the glial tumors antigens described to date aregenerally found on normal brain tissue, or are restricted tointracellular compartments, thus with limited clinical utility forantibody targeting.

Membrane antigens that have been targeted on osteosarcoma include G_(D2)(15), CD55 (16) and an as yet undefined osteosarcoma-associated antigenrecognized by the MoAbs TP-1 and TP-3 (17). However, these antigens arepresent to varying degrees on normal tissues.

Similarly the glycoprotein p30/32 coded by the MIC2 oncogene andrecognized by the monoclonal antibody O13 in the Ewing's family oftumors is expressed on normal tissues (18). In rhabdomyosarcoma, theMyoD family of oncofetal proteins is nuclear in localization (19) andtherefore inaccessible to antibody-targeted immunotherapy.

An ideal tumor antigen for targeted immunotherapy should be absent onnormal tissues and abundantly expressed on tumor cell surface. Suchtumor-specific antigens e.g. idiotypes in B cell lymphoma are rare (20).Moreover, a “generic” tumor-specific antigen expressed on tumor cells ofvarying lineage recognized by monoclonal antibodies may have broaderutility in antibody-based strategies. We describe here a noveltumor-associated antigen, recognized by a murine monoclonal antibody8H9, expressed on cell membranes of a broad spectrum of tumors ofneuroectodermal, mesenchymal and epithelial origin, with restricteddistribution on normal tissues.

SUMMARY OF THE INVENTION

This invention provides a composition comprising an effective amount ofmonoclonal antibody 8H9 or a derivative thereof and a suitable carrier.This invention provides a pharmaceutical composition comprising aneffective amount of monoclonal antibody 8H9 or a derivative thereof anda pharmaceutically acceptable carrier.

This invention also provides an antibody other than the monoclonalantibody 8H9 comprising the complementary determining regions ofmonoclonal antibody 8H9 or a derivative thereof, capable of binding tothe same antigen as the monoclonal antibody 8H9.

This invention provides a substance capable of competitively inhibitingthe binding of monoclonal antibody 8H9. In an embodiment of thesubstance, it is an antibody.

This invention provides an isolated antibody, wherein the ComplementaryDetermining Region is NYDIN, for CDR1, WIFPGDGSTQY for CDR2, QTTATWFAYfor CDR3 for the heavy chain, and RASQSISDYLH for the CDR1, YASQSIS forCDR2, QNGHSFPLT for CDR3 for the light chain. This invention furtherprovides the above antibody, wherein the other sequences are of humanorigin.

The invention also provides a composition comprising an effective amountof monoclonal antibody 8H9 or a derivative thereof and a suitablecarrier, which includes sequences as set forth in FIG. 33. In anembodiment, the sequences are mutated. This invention also provides themutated form o f 8H9, so as to reduce background and cytotoxicity. Othermutations could be established which could achieve the above-describedfunction. In a further embodiment, the antibody includes sequences asset forth in FIG. 34.

Furthermore, the invention provides a composition comprising the aboveantibodies and an isolated nucleic acid molecule encoding the antibodiesabove. This invention also provides the isolated nucleic acid moleculeabove, wherein the sequences are set forth in FIG. 33.

In addition, this invention provides a vector comprising the abovenucleic acid molecules. The invention also provides a cell comprisingthe above vector.

This invention provides an isolated scFv of monoclonal antibody 8H9 or aderivative thereof. In an embodiment, the scFv is directly or indirectlycoupled to a cytotoxic agent.

This invention provides a cell comprising 8H9-scFv. In an embodiment, itis a red cell. This invention also provides a 8H9-scFv-gene modifiedcell. This invention provides a liposome modified by 8H9-scFv.

This invention provides a method for directly kill or deliver drug, DNA,RNA or derivatives thereof to cell bearing the antigen recognized by themonoclonal antibody 8H9 or to image cells or tumors bearing said antigenusing the isolated 8H9-scFv or cell or liposome comprising the 8H9-scFv.

This invention provides a protein with about 58 kilodaltons in molecularweight, reacting specifically with the monoclonal antibody 8H9. Whenthis 58 kd protein is glycosylated, the apparent molecular weight isabout 90 kilodaltons.

This invention also provides an antibody produced by immunizing the 8H9antigen or specific portion thereof, which is immunogenic.

This invention also provides a nucleic acid molecule encoding the 8H9antigen. In addition, this invention provides a nucleic acid moleculecapable of specifically hybridizing the molecule encoding the 8H9antigen. The nucleic acid molecule includes but is not limited tosynthetic DNA, genomic DNA, cDNA or RNA.

This invention provides a vector comprising the nucleic acid moleculeencoding 8H9 antigen or a portion thereof. This invention provides acell comprising the nucleic acid molecule encoding 8H9 antigen.

This invention provides a method for producing the protein which bindsto the monoclonal antibody 8H9 comprising cloning the nucleic acidmolecule encoding the 8H9 antigen in an appropriate vector, expressingsaid protein in appropriate cells and recovery of said expressedprotein.

This invention also provides a method for production of antibody usingthe protein produced by the above method. This invention also providesantibodies produced by the above method. In an embodiment, the antibodyis a polyclonal antibody. In another embodiment, the antibody is amonoclonal.

This invention provide a method of inhibiting the growth of tumor cellscomprising contacting said tumor cells with an appropriate amount ofmonoclonal antibody 8H9 or a derivative thereof, or the antibody ofclaim produced by the expressed 8H9 antigen or a derivative of theproduced antibody thereof.

This invention provides a method of inhibiting the growth of tumor cellsin a subject comprising administering to the subject an appropriateamount of monoclonal antibody 8H9 or a derivative thereof, or theantibody produced by the expressed 8H9 antigen or a derivative thereof.

This invention provides a method for imaging a tumor in a subjectcomprising administering to the subject a labeled monoclonal antibody8H9 or labeled derivatives, or a labeled antibody produced by theexpressed 8H9 antigen or a labeled derivative. In embodiment, theantibodies or derivatives are labeled by a radioisotope.

This invention provides a method of reducing tumor cells in a subjectcomprising administering to the subject monoclonal antibody 8H9 or aderivative thereof, or a monoclonal antibody produced by the expressed8H9 antigen or a derivative thereof wherein the antibody or derivativeis coupled to a cytotoxic agent to the subject.

This invention provides a method to evaluate the tumor bearing potentialof a subject comprising measuring the expression the 8H9 antigen in thesubject, wherein the increased expression of said antigen indicateshigher tumor bearing potential of the subject.

This invention provides a transgenic animal comprising an exogenous geneencoding the 8H9 antigen. This invention also provides a knock outanimal wherein the gene encoding the 8H9 mouse analogous antigen hasbeen knocked out.

Finally, this invention provides a method to screening new anti-tumorcompound comprising contacting the above transgenic animal with thetested compound and measuring the level of expression of the 8H9 antigenin said transgenic animal, a decrease in the level of expressionindicating that the compound can inhibit the expression of the 8H9antigen and is a anti-tumor candidate.

DETAILED DESCRIPTION OF THE FIGURES

First Series of Experiments

FIG. 1. (1A) Desmoplastic small round cell tumor (10×) immunostainedwith 8H9 showing strong membrane positivity and typical histology (1B)Glioblastoma multiform stained with 8H9 showing binding to cellmembranes and fibilary stroma (1C) Embryonal rhabdomyosarcoma stainedwith 8H9 showing cell membrane reactivity (1D) Negative staining ofembryonal rhabdomyosaoma with MOPC21, an irrelevant IgG1 controlantibody

FIG. 2. Persistence of 8H9 binding to U2OS cells (2A) and NMB7 cells(2B) as studied by indirect immunofluorescence. X-axis: relativeimmunofluorescence, y-axis: hours of incubation. U2OS cells were reactedwith 8H9 and HB95, and NMB7 cells with 8H9 and 3F8. After washing, cellswere recultured and persistence of immunoreactivity of the primaryantbodies evaluated by indirect immunofluorescence using FITC-conjugatedsecondary antibody. Relative immunofluorescence of 8H9 on U2OS cellsdropped to 80% afer 48 hrs (BB95 to 11%), while that on NMB7 cellsshowed no significant drop off at 36 hrs (3F8 dropped to 39%)

FIG. 3. Effect of Pronase E on 8H9 immunoreactivity with HTB82, U2OS andNMB7 cells and on 3F8 immunoreactivity with NMB7 cells as studied byindirect immunofluorescence. X-axis: concentration of Pronase E (mg/ml);y-axis: relative immunofluorescence

Second Series of Experiments

FIG. 1. (FIG. 4 in the attached figures) 4 cycles of 3F8 and low levelHAMA response are associated with prolonged survival.

FIG. 2. (FIG. 5 in the attached figures) Improved long-term survivalafter MoAb 3F8 in patients with stage 4 NB newly diagnosed>1 year of ageat Memorial Sloan-Kettering Cancer Center. N4 to N7 are sequentialprotocols over 15 years. N4 and N5 are chemotherapy+ABMT, N6 isChemotherapy+3F8, and N7 is N6+¹³¹I-3F8.

FIG. 3. (FIG. 6 in the attached figures) Antigen modulation followingbinding to 8H9.

FIG. 4. (FIG. 7 in the attached figures) At 120 h: ¹²⁵I8H9 localized totumors (N=4) while control antibody 2C9 (mouse IgG1) remained in bloodpool/liver (N=4).

FIG. 5. (FIG. 8 in the attached figures) High tumor-tissue ratio wasspecific for ¹²⁵I-8H9 vs control MoAb ¹²⁵I-2C9 in RMS xenografts.

Third Series of Experiments

FIG. 1. (FIG. 9 in the attached figures) Reactivity of 8H9 with Ewing'ssarcoma cell lines.

Flow cytometric analysis of 8H9 binding to nine Ewing's sarcoma celllines is shown. The designation for each line is shown in the upperright corner. FL1 fluorescence of isotype (dashed black line) CD99 (thinblack line) and 8H9 (thick blackline) is shown.

FIG. 2. (FIG. 10 in the attached figures) Lack of Reactivity of 8H9 withT cells or bone marrow progenitor cells. Electronically gated Cd3+ cellsfrom peripheral blood of a normal donor (top panel) are analyzed forisotype (dashed line), CD99 (thin black line) and 8H9 (thick blackline). Electronically gated CD34+ cells from fresh human bone marrowfrom a normal donor (bottom panel are analyzed for isotype (dashed line)and 8H9 (thick black line) staining.

FIG. 3. (FIG. 11 in the attached figures) Real-time PCR analysis oft(11,22) in artificially contaminated PBMCs accurately quantifiesEWS/FII 1 transcript over up to five log dilutions of tumor. Crossingtime (x axis) is plotted vs. fluoescence (y axis) 11 a: Non-nested PCRof 10×10⁶ PBMCs contaminated from 1:10 to 1:10⁶. In the inset a linearrelationship between crossing time and log cell concentration over 4 logdilutions of tumor is shown Samples contaminated at less that 1:10⁴ showno detectable positivity in this assay. 11 b: Nested PCR of 10×10⁶ PBMCscontaminated from 1:10 to 1:10⁷. A linear relationship is observed over5 log dilutions of tumor from 1:100 to 1:10⁶.

FIG. 4. (FIG. 12 in the attached figures) Quantitative PCR analysis ofpurging demonstrates 2-3 log reduction in peripheral blood andprogenitor cells spikes with Ewing's Sarcoma cells. Cycle number (xaxis) is plotted vs. fluorescence (y axis). Experimental samples wererun with standard contaminated dilutions shown in the inset 12 a:Non-nested PCR analysis of 1×10⁶ pre-purged and post-purged non-CD34selected bone marrow from a normal healthy donor contaminated at a levelof 1:100. A two-log reduction in tumor burden is demonstrated in thepost-purged sample which shows a level of contamination at 1:10⁴. 12 b:Nested PCR analysis of pre-purged and post-purged CD34 selected cellsharvested following G-CSF mobilization from a patient with Ewing'ssarcoma. Since this patient was negative for EWS/FLI, CD34 cells werespike with Ewing's sarcoma at a level of 1:10³. A three-log reduction intumor burden is demonstrated in the post-purged sample which shows alevel of contamination at 1:10⁶. 12 c: Nested PCR analysis of prepurgedand post-purged PBMCc from a normal healthy donor buffy coatcontaminated at a level of 1:100. A greater than 3-log reduction intumor burden is demonstrated in the post-purged sample which shows alevel of contamination of less than 1:10⁶. 12 d Nested PAR analysis ofpre-purged and post-purged non PBMCs from a normal healthy donor buffycoat contaminated at a level of 1:10 ³. A 3 log reduction in tumorburden is demonstrated in the post-purged sample which shows a level ofcontamination at 1:10⁶.

FIG. 5. (FIG. 13 in the attached figures) Contamination of patientelutriated apheresis fractions is demonstrated at at level of1:10⁵-1:10⁶. Quantitative PCR analysis of apheresis fractions frompatients presenting with disseminated Ewing's sarcoma. Cycle number (xaxis) is plotted vs. flurscence y axis) Patient samples are compared tocontaminated dilutions. Patient a (top panel) shows contamination of allfractions at a level of 1:10⁵-1:10⁶. Patient B (middle panel) showscontamination in the leukocyte fraction only at a level of approximately1:10⁶, Patient C (bottom panel) shows contamination in several fractionsat a similar level.

FIG. 6. (FIG. 14 in the attached figures) Progenitor CFU capability isnot affected by 8H9 based purging. Colony formy units from CD34 selectedcells from bone marrow from a normal healthy donor (x axis) are plottedfor pre- and post purged samples.

FIG. 7. (FIG. 15 in the attached figures) OKT3 mediated T cellproliferation is unchanged after purging when compared to prepurgedproliferation. T cells from normal healthy donor buffy coat wereevalauted for [³H] Thymidine uptake as a measure of T cell proliferationwith a decreasing concentration of OKT3. Uptake is measured as countsper million y axis) and is plotted vs. OKT3 concentration for pre-purged(solid square), and post purged (solid circle).

Fourth Series of Experiments

FIG. 1. (FIG. 16 in the attached figures) DSRCT (40×) demonstrating cellmembrane and stromal reactivity with 3F8

FIG. 2. (FIG. 17 in the attached figures) DSRCR (40×) showing strong,homogeneous, cell membrane and stromal reactivity with 8H9

Fifth Series of Experiments

FIG. 1. FIG. 18 in the attached figures) Inhibition of 8EP9 byanti-idiotype 2E9 by FACS analysis. 18A: Staining of LAN-1 neuroblastomacells with 5 ug/ml of 8H9 (shaded peak) was not inhibited at lowconcentration of 2E9 (2 ug/ml, solid line), but almost completely atconcentration of 10 ug/ml (dotted line) superimposable with the negativeantibody control (solid line). 18B: Staining of LAN-1 neuroblastomacells with 5 ug/ml of 3F8 (anti-GD2, shaded peak) was not inhibited byany concentrations (2 ug/ml, solid line, or 200 ug/ml, dotted line) of2E9. 18C Staining of HTB-82 rhabdomyosarcoma cells with 5 ug/ml of 8H9(grey peaks was not inhibited at low concentration (2 ug/ml solid line),but completely at 10 ug/ml of 2E9 (solid line) superimposable withnegative antibody control (black peak).

FIG. 2. (FIG. 19 in the attached figures) SDS-PAGE lanes a and b) andWestern blot (c and d) of 8H9 scFv-Fc. H=heavy chain of 8H9, L=lightchain of 8H9, scFv-Fc=chimeric fusion protein between 8H9 scFv and thehuman, 1-CH2-CH3 domain. With 2-mercaptothanol: lanes a, b and c. Nativegel: lane d. SDS-PAGE was stained with Comassie Blue; western blot with2E9 anti-idiotypic antibody.

FIG. 3. (FIG. 20 in the attached figures) FACS analysis of 8h9-scFv-Fcstaining of HTB82 rhabdomyosarcoma cells. 20A Immunofluorescenceincreased with concentrations of 8H9-scFv-Fc (1, 5, 25, 125 ug/ml),shaded peak is no antibody control. 20B: Cell staining (5 ug/ml of8H9-scFv-Fc, thin solid line) was completely inhibited (thick solidline) at 1 ug/ml of anti-idiotypic antibody 2E9, shaded peak is noantibody control.

FIG. 4. (FIG. 21 in the attached figures) Immunoscintigraphy of humantumors using ¹²⁵I-labeled 8H9 scFv-Fc. Mice xenografted with human LAN-1neuroblastoma received retroorbital injections of 25 uCi of ¹²⁵I-labeledantibody. 24 h, 48 h and 7 days after injection, the animals wereanesthesized and imaged with a gamma camera.

FIG. 5. (FIG. 22 in the attached figures) Blood clearance of¹²⁵I-labeled chimeric 8H9 and ¹²⁵I-native 8H9. Mice xenografted withhuman LAN-1 neuroblastoma received retroorbital injections of¹²⁵I-labeled antibody. Percent injected dose/gm of serial blood sampleswere plotted over time.

Sixth Series of Experiments

FIG. 1. (FIG. 23 in the attached figures) Anti-idiotype affinityenrichment of producer lines. Producer lines were stained withanti-idiotypic MoAb 2E9 before (shaded peak, A and B), and after first(dotted line peak, A) and second (thick solid line, A) affinitypurification, and after first (dotted line, B) and second (solid line B)subcloning, showing improved scFv expression. By FACS the indicator lineK562 showed improved scFv expression after first (dotted line, C) andsecond (thick solid line, C) affinity purification of the producer line,and subsequent first (dotted line, C) and second (thick solid line, D)subcloning of the producer line, when compared to unpurified producerlines (shaded peaks, C and D), consistent with improvement in genetransduction efficiency. The thin solid line curves in each figurerepresents nonproducer line (A and B) or uninfected K562 (C and D).

FIG. 2. (FIG. 24 in the attached figures) Flow cytometry analysis ofscFv expression. Cultured 8H9-scFv-CD28-gene-modified lymphocytes wereassayed for their scFv expression using anti-idiotypic MoAb 2E9 (solidcurves) and control rat IgG1 MoAb as control (shaded histograms) fromday 13 through day 62. Although a substantial proportion of cells werepositive by day 13, they became homogeneous by day 21 and persisted tillday 62, when the overall mean fluorescence appeared to decrease.

FIG. 3. FIG. 25 in the attached figures) In vitro expansion of8H9-scFv-CD2{tilde over (8)}-gene-modified primary human lymphocytes.Clonal expansion was expressed as a fraction of the initial viable cellnumber. IL-2 (100 U/ml) was added after retroviral infection and waspresent throughout the entire in vitro culture period. Short bars depictthe days when soluble anti-idiotypic antibody 2E9 (3-10 ug/ml) waspresent in the culture.

FIG. 4. FIG. 26 in the attached figures) Cytotoxicity against tumor celllines: 8H9-scFv-CD2{tilde over (8)}-gene-modified lymphocytes from day56 of culture (scFv-T) were assayed by ⁵¹Cr release assay in thepresence or absence of 8H9 (50 ug/ml final concentration) as an antigenblocking agent. Control lymphocytes (LAK) from the same donor but notgene-modified, were cultured under the same conditions as thegene-modified cells. 26A: NMB-7 neuroblastoma. 26B: LAN-1 neuroblastoma.26C: HTB-82 rhabdomyosarcoma. 26D: Daudi lymphoma.

FIG. 5. (FIG. 27 in the attached figures) Suppression ofrhabdomyosarcoma tumor growth in SCID mice. Human rhabdomyosarcomaHTB-82 was strongly reactive with 8H9, but not with 5F11 (anti-GD2)antibodies. Experimental group: 8H9-scFv-CD2{tilde over(8)}-gene-modified human lymphocytes (solid circles). Control groups: nocells+2E9 (open circles), cultured unmodified lymphocytes (LAK)+2E9(open triangles), or 5F11scFv-CD2{tilde over (8)}-modifiedlymphocytes+1G8 [rat anti-5F11 anti-idiotype MoAb] (solid triangles).

Seventh Series of Experiments

FIG. 1. (FIG. 28 in the attached figures) Sequential imaging of nudemouse bearing RMS xenograft 24, 48 and 172 h after injection with 4.4MBq ¹²⁵I-8H9 as compared to a RMS xenograft-bearing mouse imaged 172 hafter injection with 4 4 MBq ¹²⁵I-2C9.

FIG. 2. FIG. 29 in the attached figures) Blood kinetics of ¹²⁵I-8H9 innude mice with RMS xenografts. Error bars represent SEM.

FIG. 3. (FIG. 30 in the attached figures) Comparison of biodistributionof ¹²⁵I-8H9 at 24, 48 and 172 h after injection in xerograft and normaltissues.

FIG. 4. (FIG. 31 in the attached figures) Comparison of biodistributionof ¹²⁵I-8H9 with that of the nonspecific anticyokeratin MoAb ¹²⁵I-2C9(solid bars) in xenografts and normal tissues.

FIG. 5. (FIG. 32 in the attached figures) Anti tumor effect on RMSxenografts: ¹³¹I-8H9 versus negative control MoAb¹³¹I-3F8. Each mousereceived 18.5 MBq radiolabeled MoAb (5 mice per group).

FIG. 33: 8H9 scFv gene sequence (sense and complementary). Complementarydetermining regions (CDR) are marked in boxes in the following order:CDR-1 (HC, heavy chain), CDR-2 (HC), CDr-3 (HC), CDR-1 (LC, lightchain), CDR-2 (LC), CDR-3 (LC).

FIG. 34: Gene and amino acid sequences of 8 H9scFv is depicted. Mutated8H9 scFv carries the following site-directed mutagenesis (VH: K13E andVL: R18Q, R45Q, K103E, K107E) to decrease PI from 6.4 to 4.8, and netcharge from −1 to −9, a strategy to decrease nonspecific normal tissueadherence.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition comprising an effective amount ofmonoclonal antibody 8H9 or a derivative thereof and a suitable carrier.This invention provides a pharmaceutical composition comprising aneffective amount of monoclonal antibody 8H9 or a derivative thereof anda pharmaceutically acceptable carrier. In an embodiment of the abovecomposition, the derivative is a scFv. In a separate embodiment, theantibody is an antibody-fusion construct In another embodiment, theantibody is an scFvFc.

This invention provides an isolated antibody, wherein the ComplementaryDetermining Region is NYDIN, for CDR1, WIFPGDGSTQY for CDR2, QTTATWFAYfor CDR3 for the heavy chain, and RASQSISDYLH for the CDR1, YASQSIS forCDR2, QNGHSFPLT for CDR3 for the light chain. This invention furtherprovides the above antibody, wherein the other sequences are of humanorigin.

The invention also provides a composition comprising an effective amountof monoclonal antibody 8H9 or a derivative thereof and a suitablecarrier, which includes sequences as set forth in FIG. 33. In anembodiment, the sequences are mutated. This invention also provides themutated form of 8H9, so as to reduce background and cytotoxicity. Othermutations could be established which could achieve the above-describedfunction. In a further embodiment, the antibody includes sequences asset forth in FIG. 34.

Furthermore, the invention provides a composition comprising the aboveantibodies and an isolated nucleic acid molecule encoding the antibodiesabove. This invention also provides the isolated nucleic acid moleculeabove, wherein the sequences are set forth in FIG. 33.

This invention also provides a peptide or polypeptide comprising: NYDIN,WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, QNGHSFPLT alone or incombination thereof. The peptide or polypeptide functions similarly tothe 8H9 antibody and therefore, for the below uses, this peptide orpolypeptide may be used similarly. In an embodiment, the inventionprovides an isolated nucleic acid encoding the above peptide.

In addition, this invention provides a vector comprising the abovenucleic acid molecules. The invention also provides a cell comprisingthe above vector. This invention further provides cells expressing the8H9 or derivative of 8H9 antibodies.

This invention provides an antibody other than the monoclonal antibody8H9 comprising the complementary determining regions of monoclonalantibody 8H9 or a derivative thereof, capable of binding to the sameantigen as the monoclonal antibody 8H9.

This invention also provides a substance capable of competitivelyinhibiting the binding of monoclonal antibody 8H9. In an embodiment, thesubstance is an antibody. In another embodiment, the substance is apeptide.

This invention further provides an isolated single chain antibody of 8H9or a derivative thereof. This invention also provides an isolated scFvof monoclonal antibody 8H9 or a derivative thereof. Single chainantibodies or derivatives are known in the antibody field. In anembodiment, the scFv is directly or indirectly coupled to a cytotoxicagent. In a further embodiment, the scFv is linked to a first proteincapable of binding to a second protein which is coupled to a cytotoxicagent. Same rationale applies to the imaging uses of the 8H9 monoclonalantibody or its derivative. In the case of imaging, instead of acytotoxic agent, the antibody or its derivative will be coupled to animaging agent. Both cytotoxic or imaging agents are known in the art.

This invention provides a cell comprising 8H9-scFv. In an embodiment,the cell is a red cell. This invention also provides a 8H9-scFv-genemodified cell.

This invention also provides a liposome modified by 8H9-scFv. It is oneintention of the disclosure to deliver 8H9 via liposomes or via otherdelivery technologies.

This invention provides a method to directly kill, or deliver drug, DNA,RNA or derivatives thereof to cell bearing the antigen recognized by themonoclonal antibody 8H9 or to image cells or tumors bearing said antigenusing the isolated 8H9-scFv or 8H9-scFv modified cell or liposome.

This invention provides a protein with about 58 kilodaltons in molecularweight, reacting specifically with the monoclonal antibody 8H9. Whenthis protein is glycosylated, the apparent molecular weight is about 90kilodaltons.

This invention provides an antibody produced by immunizing the expressed8H9 antigen or specific portion thereof. The specific portion is theregion which is recognized by 8H9. Another region is the region whichperforms its specific function.

This invention provides a nucleic acid molecule encoding 8H9 antigen.

This invention provides a nucleic acid molecule capable of specificallyhybridizing the nucleic acid molecule which encodes the 8H9 antigen. Thenucleic acid molecule includes but is not limited to synthetic DNA,genomic DNA, cDNA or RNA.

This invention also provides a vector comprising the nucleic acidmolecule encoding the 8H9 antigen or a portion thereof. The portioncould be a functional domain of said antigen. This invention provides acell comprising the nucleic acid molecule encoding the 8H9 antigen.

This invention provides a method for producing the protein which bindsto the monoclonal antibody 8H9 comprising cloning the nucleic acidmolecule which encodes the 8H9 antigen in an appropriate vector,expressing said protein in appropriate cells and recovery of saidexpressed protein.

This invention provides a method for production of antibody using theexpressed 8H9 antigen or the portion which is immunogenic. Thisinvention also provides an antibody produced by the above describedmethod. In an embodiment, the antibody is polyclonal. In anotherembodiment, the antibody is a monoclonal.

This invention provides a method of inhibiting the growth of tumor cellscomprising contacting said tumor cells with an appropriate amount ofmonoclonal antibody 8H9 or a derivative thereof, or the antibodyproduced using the expressed 8H9 antigen or a derivative thereof.

This invention provides a method of inhibiting the growth of tumor cellsin a subject comprising administering to the subject an appropriateamount of monoclonal antibody 8H9 or a derivative thereof, or theantibody produced using the expressed 8H9 antigen or a derivativethereof.

This invention provides a method for imaging a tumor in a subjectcomprising administering to the subject a labeled monoclonal antibody8H9 or a labeled derivatives, or a labeled antibody produced using theexpressed 8H9 antigen or a labeled derivative. In an embodiment, theantibody or the derivative is labeled with radioisotope.

This invention provides a method of reducing tumor cells in a subjectcomprising administering to the subject monoclonal antibody 8H9 or aderivative thereof, or a monoclonal antibody produced using theexpressed 8H9 antigen or a derivative thereof wherein the antibody orderivative is coupled to a cytotoxic agent to the subject. In anembodiment, the coupling to a cytotoxic agent is indirect. In anotherembodiment, the coupling is first directly linking the antibody orderivative with a first protein which is capable of bind to a secondprotein and the second protein is covalently couple to a cytotoxicagent. In a further embodiment, the cytotoxic agent is a radioisotope.

This invention also provides a method to evaluate the tumor bearingpotential of a subject comprising measuring the expression the 8H9antigen in the subject, wherein the increased expression of said antigenindicates higher tumor bearing potential of the subject.

This invention provides a transgenic animal comprising an exogenous geneencoding the 8H9 antigen. The transgenic animal may also carried anknock out gene encoding the 8H9 mouse analogous antigen. In anembodiment, it is a transgenic mouse.

This invention provides a method to screening new anti-tumor compoundcomprising contacting the transgenic animal with the tested compound andmeasuring the level of expression of the 8H9 antigen in said transgenicanimal, a decrease in the level of expression indicating that thecompound can inhibit the expression of the 8H9 antigen and is aanti-tumor candidate.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

EXPERIMENTAL DETAILS

First Series of Experiments

Monoclonal Antibody 8H9 Targets a Novel Cell Surface Antigen Expressedby a Wide Spectrum of Humans Solid Tumors

Tumor-restricted surface antigens may be targets for diagnosis andimmune-based therapies. Monoclonal antibody 8H9 is a murine IgG1hybridoma derived from the fusion of mouse myeloma SP2/0 cells andsplenic lymphocytes from BALB/c mice immunized with human neuroblastoma.By immunohistochemistry, 8H9 was highly reactive with human braintumors, childhood sarcomas, neuroblastomas and less so withadenocarcinomas. Among primary brain tumors, 15/17 glioblastomas, 3/4mixed gliomas, 4/11 oligodendrogliomas, 6/8 astrocytomas, 2/2meningiomas, 3/3 schwannomas, 2/2 medulloblastomas, 1/1 neurofibroma,1/2 neuronoglial tumors, 2/3 ependymomas and 1/1 pineoblastoma weretested positive. Among sarcomas, 21/21 Ewing's/PNET, 28/29rhabdomyosarcoma, 28/29 osteosarcomas, 35/37 desmoplastic small roundcell tumors, 2/3 synovial sarcomas, 4/4 leiomyosarcomas, 1/1 malignantfibrous histiocytoma and 2/2 undifferentiated sarcomas tested positivewith 8H9. 87/90 neuroblastomas, 12/16 melanomas, 3/4 hepatoblastomas,7/8 Wilm's tumors, 3/3 rhabdoid tumors and 12/27 adenocarcinomas alsotested positive. In contrast 8H9 was nonreactive with normal humantissues including bone marrow, colon, stomach, heart, lung, muscle,thyroid, testes, pancreas, and human brain (frontal lobe, cerebellum,pons and spinal cord). Reactivity with normal cynomolgus monkey tissuewas similarly restricted. Indirect immunofluorescence localized theantigen recognized by 8H9 to the cell membrane. The antigen isproteinase-sensitive and is not easily modulated off cell surface. 8H9immuno-precipitated a 58 kD band following N-glycanase treatment, mostlikely a protein with heterogeneous degree of glycosylation. This novelantibody-antigen system may have potential for tumor targeting.

Monoclonal antibodies such as 3F8 (1) and 14.18 (2) against G_(D2) inneuroblastoma, M195 against CD33 in acute leukemia (3), anti-HER2antibodies in breast cancer (4) and anti-CD20 antibodies in lymphoma (5)have shown efficacy in recent clinical trials. The prognosis in glialbrain tumors and metastatic mesenchymal and neuroectodermal tumorsremains dismal despite innovations in chemotherapy and radiationtherapy. Immunotherapy may offer new possibilities for improving theoutcome in these patients.

Tumor antigens expressed on cell membrane are potential targets inimmunotherapy. Examples of tumor antigens expressed on glial tumorsinclude neural cell adhesion molecules (6), gangliosides such as G_(D2)and G_(M2) (7), and neurohematopoeitic antigens (8). Recentinvestigations have focused on growth factor receptors as immunetargets, in particular type III mutant epidermal growth factor receptor(EGFRvIII) which has been shown to be expressed on 50% of glial braintumors (9). Notwithstanding the universal expression of NCAM by neuronalcells, two clinical studies have utilized anti-NCAM antibodies inpatients. MAb UJ13A was shown to accumulate in gliomas by virtue ofdisruption of blood brain barrier locally (10) and another antibody,ERIC-1 was used in a therapeutic setting in resected glioma cavitieswith some clinical benefit (11)

Recent studies have targeted immunotherapy to extracellular matrixaround tumor cells. Tenascin has been reported to be expressed in 50-95%of glial brain tumors as well as on mesenchymal tumors, carcinomas andnormal human glial, liver and kidney cells (12). Anti-tenascinmonoclonal antibodies 81C6 (13) and BC-2 and BC-4 (14) administeredintra-cavity have recently been reported to show efficacy in thetreatment of patients with malignant gliomas. However, since theseantigens are also present to varying degrees on normal human neural andnon-neural cells, their clinical utility would depend on theiroverexpression by brain tumors when compared to normal tissues. With theexception of EGFRvIII, the glial tumors antigens described to date aregenerally found on normal brain tissue, or are restricted tointracellular compartments, thus with limited clinical utility forantibody targeting.

Membrane antigens that have been targeted on osteosarcoma include G_(D2)(15), CD55 (16) and an as yet undefined osteosarcoma-associated antigenrecognized by the MoAbs TP-1 and TP-3 (17). However, these antigens arepresent to varying degrees on normal tissues. Similarly the glycoproteinp 30/32 coded by the MIC2 oncogene and recognized by the monoclonalantibody O13 in the Ewing's family of tumors is expressed on normaltissues (18). In rhabdomyosarcoma, the MyoD family of oncofetal proteinsis nuclear in localization (19) and therefore inaccessible toantibody-targeted immunotherapy.

An ideal tumor antigen for targeted immunotherapy should be absent onnormal tissues and abundantly expressed on tumor cell surface. Suchtumor-specific antigens e.g. idiotypes in B cell lymphoma are rare (20).Moreover, a “generic” tumor-specific antigen expressed on tumor cells ofvarying lineage recognized by monoclonal antibodies may have broaderutility in antibody-based strategies. We describe here a noveltumor-associated antigen, recognized by a murine monoclonal antibody8H9, expressed on cell membranes of a broad spectrum of tumors ofneuroectodermal, mesenchymal and epithelial origin, with restricteddistribution on normal tissues.

MATERIALS AND METHODS

Tumor and Normal Tissue Samples

Frozen tumors from 330 patients with neuroectodermal, mesenchymal andepithelial neoplasia were analyzed. All diagnoses of tumor samples wereconfirmed by hematoxylin and eosin assessment of paraffin-embeddedspecimens. 15 normal human tissue samples and 8 normal cynomolgus monkeytissue samples obtained at autopsy were also analyzed.

Cell Lines

Human neuroblastoma cell lines LA-N-1 was provided by Dr. Robert Seeger,Children's Hospital of Los Angeles, Los Angeles, Calif. Humanneuroblastoma cell lines LA-15-N, LA-66-N, LA-5S, LA-19-S and LA-19-Nwere provided by Dr. Robert Ross (Fordham University, N.Y.) and IMR 32and NMB7 by Dr. Shuen-Kuei Liao (McMaster University, Ontario, Canada).Breast carcinoma cell lines SW480 and ZR75-1 were provided by Dr. S.Welt (Memorial Sloan-Kettering Cancer Center, NY) and the melanoma lineSKMel28 by Dr. P. Chapman (Memorial Sloan-Kettering Cancer Center, NY).Neuroblastoma cell lines SKNHM, SKNHB, SKNJD, SKNLP, SKNER, SKNMM, SKNCHand SKNSH, rhabdomyosarcoma cell line SKRJC and Ewing's/PNET cell linesSKPPR, SKPRT and SKNMC were derived from patients with metastaticdisease treated at MSKCC. The following cell lines were purchased fromAmerican Type Culture Collection, Bethesda, Md.: melanoma cell linesHTB63 and HTB67, rhabdomyosarcoma cell line HTB82, small cell lungcancer cell line HTB 119, acute T-leukemia cell line Jurkat,glioblastoma multiformed cell line Glio72, breast cancer cell line HTB22, colon carcinoma cells line SK Co-1, Hela, embryonal kidney 293, andosteosarcoma cell lines CRL1427, HTB86 and HTB 96. All cell lines weregrown at 37° C. in a 6% CO₂ incubator using standard culture medium,which consisted of RPMI 1640 medium supplemented with 10% bovine calfserum, 2 mM glutamine, penicillin (100 IU/ml) and streptomycin (100μg/ml). Normal human hepatocytes were purchased from Clonetics, SanDiego, Calif. and processed immediately upon delivery. Normal humanmononuclear cells were prepared from heparinized bone marrow samples bycentrifugation across a Ficoll-Hypaque density separation gradient. EBVlymphoblastoid cell lines were derived from human mononuclear cells.

Monoclonal Antibody

Female BALB/c mice were hyperimmunized with human neuroblastomaaccording to previously outlined methods (21). Lymphocytes derived fromthese mice were fused with SP2/0 mouse myeloma cells line. Clones wereselected for specific binding on ELISA. The 8H9 hybridoma secreting anIgG₁ monoclonal antibody was selected for further characterization aftersubcloning.

Immunohistochemical Studies

Eight μm cryostat frozen tumor sections were fixed in acetone and washedin PBS. Immunohistochemical studies were performed as describedpreviously (22). Endogenous peroxidases were blocked in 0.3% H₂O₂ inPBS. Sections were incubated in 10% horse serum (Gibco BRL,Gaithersburg, Md.) after blocking with avidin and biotin. Incubationwith purified 8H9 (2 μg/ml) in PBS was carried out at room temperaturefor 1 hour. An IgG1 myeloma was used as a control (Sigma Chemical, StLouis Mo.). Sections were incubated with a secondary horse anti-mousebiotinylated antibody (Vector Laboratories, Burlingame, Calif.) followedby incubation with ABC complex (Vector) and developed with Vector VIPperoxidase substrate or DAB peroxidase substrate kit (Vector). A 10%hematoxylin counterstain for 4 minutes was used. Staining was graded aspositive or negative and homogeneous or heterogeneous reactivity noted.

Indirect Immunofluorescence

1 million target cells were washed in PBS and then spun at 180×g for 5min. The pellets were then reacted with 100 μl of 15 μg/ml 8H9 at 4° C.for 1 hour. After washing the cells with PBS they were allowed to reactwith 100 μl FITC-conjugated goat F (ab′)₂ anti-mouse IgG+IgM, (BiosourceInternational, Camarillo, Calif.) at 4° C. Flow cytometric analysis wasperformed using FACSCalibur Immunocytometer (Becton-DickinsonImmunocytometry Systems, San Jose, Calif.).

In order to study loss of antigen after binding to 8H9, 10⁶ NMB7 andU2OS cell pellets were prepared as above and reacted with 100 μl each of15 μg/ml of 8H9 or the anti-HLA A,B,C antibody, HB-95 (American TypeCulture Collection, Bethesda, Md.) at 4° C. for 1 hour. NMB7 cells werealso similarly reacted with the anti-G_(D2) monoclonal antibody 3F8.After washing with PBS, cells were cultured at 37° C. in standardculture medium for 0, 1, 2, 4, 8, 12, 24, 36 and 48 h. They were thenreacted with FITC-conjugated secondary antibody goat F(ab′)₂ anti-mouseIgG+IgM, (Biosource International, Camarillo, Calif.) at 4° C. Flowcytometric analysis was performed. Geometric mean immunofluorescence wascompared to that of control cells incubated for similar time intervalsin standard culture medium in the absence of MoAbs, and thenimmunostained with HB-95 (U2OS) or 3F8 (NMB7).

Antigen sensitivity to proteinase was tested by incubating 0.5×10⁶ ofHTB82, U2OS and NMB7 cells at 37° C. for 30 minutes in RPMI withincreasing concentrations of neutral proteinase, Pronase E fromstreptomyces griseus (L Merck, Darmstadt, Germany) After washing, cellswere stained with 8H9 or 3F8 and studied by indirect immunofluorescence.

Immunoprecipitation

Immunoprecipitation was carried out using a modification of the standardtechnique. (23) 8H9-positive cell lines (NMB7, LAN-1, HTB82, U2OS, HELA,293) and 8H9-negative cell lines (Jurkat, HTB119) were used. 2×10⁷viable cells were washed in TBS (0.05 M Tris-HCl, pH 8, with 0.15 MNaCl) and incubated with 10 U lactoperoxidase (Sigma) 100 ul of 100 U/mlin TBS, 1 mCi ¹²⁵I (2.7 ul) and 1/6000 dilution of 30% hydrogen peroxidefor 5 min at 20° C. Five units of lactoperoxidase (50 ul) and the samedilution of hydrogen peroxide (50 ul) were added every 3 min with mixingfor a total of 3 times. The cells were washed extensively in TBScontaining 2 mg/ml of NaI. The iodinated cells were washed three timesin TBS, lysed on ice (30 min) in 500 ul of modified RIPA buffer (0.01 MTris-HCl, pH 7.2, 0.15 M NaCl, 1% sodium deoxycholate, 1% Nonidet P-40,0.1% sodium dodecyl sulfate (SDS), 0.01 M EDTA) containing proteaseinhibitors (1 mM PMSF, 50 ug/ml Bestatin, 2 ug/ml Aprotinin, 0.5 ug/mlLeupeptin, 0.7 ug/ml Pepstatin, 10 ug/ml E-64). The lysates wereclarified by centrifugation at 15,000 rpm for 5 min at 4° C., thenincubated with 1 mg of 8H9 or IgG1 control antibody for 16 hr at 4° C.with mixing. The antigen-antibody complex was collected by adsorptiononto 100 ul Protein G-sepharose beads (Sigma) for 6 hr at 4° C. Theimmune complex immobilized on Protein G was washed three times withmodified RIPA buffer, and then washed once with RIPA buffer containing 1M NaCl, and then twice with modified TNN buffer (0.05 M Tris-HCl, pH 8,0.15 M NaCl, 0.05% Nonidet P-40). Bound proteins were removed by elutionwith SDS-sample buffer and analyzed by 7.5% SDS-PAGE, followed byautoradiography. Deglycosylation of the radiolabeled antigen was carriedout on the protein G sepharose using N-glycanase (Glyco, Novato, Calif.)and O-glycanase (Glyco) according to manufacturers' instructions.Molecular weight was estimated using Quantity One software from BioRadInc. (Hercules, Calif.).

RESULTS

Immunohistochemical Studies

Frozen sections from 330 tumors with histologically confirmed diagnosesof cancer were analyzed for immunoreactivity with mAb 8H9 (Tables 1, 2).15 histologically normal human tissues and 8 normal monkey tissues werealso analyzed (Table 3). TABLE 1 8H9 reactivity: neuroectodermal Tumors8H9 Tumors No. positive % Neuroblastoma Brain Tumors 90 87 97 1. GlialTumors Glioblastoma multiforme 17 15 88 Mixed Glioma 4 3 —Oligodendroglioma 11 4 36 Astrocytoma 8 6 75 Ependymoma 3 2 — 2.Primitive PNET Medulloblastoma 2 2 — 3. Mixed Neuronoglial tumor 2 1 —4. Other Schwannoma 3 3 — Meningioma 2 2 — Neurofibroma 1 1 —Pineoblastoma 1 1 — Melanoma 16 12 75 Ewing's Family of tumors 21 21 100TOTAL 181 160 88

TABLE 2 8H9 reactivity: mesenchymal, epithelial and other tumors 8H9Tumors No. Reactive % A. Mesenchymal Rhabdomyosarcoma 29 28 97Osteosarcoma 29 28 97 Desmoplastic small round 37 35 95 cell tumorLeiomyosarcoma 4 4 — Synovial sarcoma 3 2 — Malignant fibroushistiocytoma 1 1 — Undifferentiated sarcoma 2 2 — Fibrosarcoma 1 0 TOTAL106 100 94 B. Epithelial Breast 12 4 33 Bladder 4 1 — Adrenal 3 1 —Stomach 1 1 — Prostate 2 1 — Colon 1 1 — Lung 1 1 — Endometrium 1 1 —Cervix 1 0 — Renal 1 1 — TOTAL 27 12 44 Epithelial tumors summary No.Slide Date Diagnosis 8H9 1 7251 Mar. 11, 1998 Breast Ca neg 2 7279 Mar.13, 1998 Breast Ca neg 3 7282/ Mar. 13, 1998; Breast Ca neg 7601 Oct. 5,1998 4 7722 Oct. 21, 1998 Breast Ca NE(no cells) 5 7261 Mar. 11, 1998Breast Ca pos 6 6388 Aug. 26, 1998 Breast Ca pos 7 6493 Oct. 11, 1998Breast Ca neg 8 6498 Oct. 11, 1998 Breast Ca neg 9 6499 Oct. 11, 1998Breast Ca neg 10 6492 Oct. 11, 1998 Breast Ca neg 11 6376 Aug. 26, 1996Breast Ca pos 12 6488 Oct. 11, 1998 Bladder Ca neg 13 6489 Oct. 11, 1998Bladder Ca weakly + 14 6490 Oct. 11, 1998 Bladder Ca neg 15 6491 Oct.11, 1998 Bladder Ca neg 16 6441 Sept. 30, 1998 Lung Ca pos 17 6503 Oct.11, 1998 Prostate Ca neg 18 6504 Oct. 11, 1998 Prostate Ca pos 19 6501Oct. 11, 1998 Cervix Ca neg 20 6502 Oct. 11, 1998 Uterine Ca pos 21 7717Oct. 21, 1998 Adrenal Ca ne (necrotic) 22 7250 Mar. 11, 1998 Adrenal Caneg 23 7207 Nov. 18, 1998 Renal Ca pos 24 6505 Oct. 11, 1998 Stomach Capos 25 7886 Feb. 22, 1999 Adrenal Ca pos Total Evaluable 8H9 pos. Breast11 10 3 of 10 Bladder 4 4 1 of 4 Prostate 2 2 1 of 2 Adrenal 3 2 1 of 2Renal 1 1 1 of 1 Stomach 1 1 1 of 1 Uterine 1 1 1 of 1 Cervix 1 1 0 of 1Lung 1 1 1 of 1 TOTAL 25 23 10 of 23 C. Other tumors Tumors No. 8H9reactive % Hepatoblastoma 4 3 — Wilm's tumor 8 7 — Rhabdoid tumor 3 3 —Paraganglioma 1 1 — TOTAL 16 14 88

TABLE 3 8H9 reactivity in normal human and cynomolgus monkey tissuesTissues 8H9 reactivity A. Human Frontal lobe Negative Pons NegativeSpinal cord Negative Cerebellum Negative Lung Negative Heart NegativeSkeletal muscle Negative Thyroid Negative Testes Negative Pancreascytoplasmic staining* Adrenal cortex cytoplasmic staining* Livercytoplasmic staining* Sigmoid colon Negative Bone Marrow Negative KidneyNegative B. Cynomolgus monkey Cerebellum Negative Frontal Lobe NegativeOccipital Cortex Negative Brainstem Negative Liver cytoplasmic stainingStomach Negative Adrenal Cortex cytoplasmic staining Kidney Negative*non-specific background

Heterogenous, non-specific cytoplasmic staining was noticed in normalhuman pancreas, stomach, liver and adrenal cortex which was diminishedwhen 8H9 F(ab′)₂ fragments were used instead of the whole antibody forimmunostaining (data not shown). None of the other human tissues showedreactivity with 8H9. In particular normal human brain tissue sectionsincluding frontal lobe, spinal cord, pons and cerebellum were completelynegative. Normal tissues from cynomolgus monkey also demonstratedsimilarly restricted reactivity with nonspecific staining observed instomach and liver. (Table 3)

The majority of neuroectodermal and mesenchymal tumors tested showedpositive reactivity with 8H9, epithelial tumors to a lesser extent. 8H9immunoreactivity was seen in a characteristic, homogeneous, cellmembrane distribution in 286 of the 330 (87%) tumor samples examined.(FIG. 1). 88% of neuroectodermal tumors, 94% of mesenchymal tumors and44% of epithelial tumors tested positive with 8H9. (Tables 2, 3)

Indirect Immunofluorescence

8H9 immunoreactivity in 35 neuroblastoma, melanoma, rhabdomyosarcoma,small cell lung cancer, osteosarcoma, glioblastoma, leukemia, breastcancer and colon cancer cell lines was tested using indirectimmunofluorescence. Moderate to strong cell membrane reactivity with 8H9was detected in 16/16 neuroblastoma cell lines, 3/3 melanoma cell lines,2/2 rhabdomyosarcoma cell lines, 1/1 glioblastoma multiformed cell line,3/3 breast cancer cell lines and 1/1 colon cancer cell lines studied. 2of 3 Ewing's/PNET cell lines, and 2 of the 3 osteosarcoma cell lineswere strongly positive while the others showed weak positivity. Thesmall cell lung cancer cell line tested negative with 8H9 as did JurkatT-ALL cell line and EBV transformed lymphoblastoid cells. Normal humanbone marrow mononuclear cells and hepatocytes had no reactivity with8H9. (Table 4) In the neuroblastoma cell lines studied, indirectimmunofluorescence with 8H9 was weaker (mean fluorescence: 73.73;negative control: 3.95) when compared to the anti-G_(D2) antibody 3F8(mean fluorescence: 249.95).

8H9 binding to U2OS as detected by indirect immunofluorescence did notdiminish significantly after 48 hr of incubation at 37° C. During thesame period, binding to the anti-HLA antibody HB-95 diminished by 89%.Similarly there was no significant loss of 8H9 binding to NMB7 cells.whereas 3F8 binding diminished by 61%. (FIG. 2)

There was a pronase dose-dependent reduction in reactivity with 8H9 with75-85% loss of immunofluorescence achieved at a final Pronaseconcentration of 0.3 mg/ml FIG. 3). There was no appreciable loss ofreactivity with 3F8 on NMB7 cells. Furthermore, the 8H9 antigen was notsensitive to neuraminidase or phosphatidyl-inositol specificphospholipase C (data not shown). TABLE 4 8H9 reactivity with cell linesby indirect immunofluorescence Cell line 8H9 Reactivity 1. NeuroblastomaLA-N-1 positive NMB7 positive LA-1-15-N positive LA-1-66N positive IMR32positive LA-1-19N positive LA-1-5S positive LA-1-19S positive SKNHMpositive SKNSH positive SKNHB positive SKNJD positive SKNLP positiveSKNMM positive SKPCH positive SKNER positive 2. Melanoma HTB63 positiveHTB67 positive SKMel28 positive 3. Rhabdomyosarcoma HTB 82 positiveSKRJC positive 4. Small cell lung cancer HTB119 negative 5. OsteosarcomaCRL1427 positive HTB96 positive HTB86 positive 6. Ewing's/PNET SKPPRpositive SKPRT positive SKNMC positive 7. Glioblastoma Glio72 positive8. Carcinoma Breast ZR75-1 positive SW480 positive HTB22 positive 9.Carcinoma Colon SKCo-1 positive 10. Leukemia Jurkat negative 11. Normalhuman cells negative Bone marrow negative Hepatocytes negative 12. EBVlymphoblastoid cells negativeImmunoprecipitation:

8H9 immunoprecipitated a broad band centered around 90 kD from all the8H9-positive cell lines (HTB82, NMB7, LAN1, U2OS, Hela, 293), whetherusing native or reducing (2ME) conditions (data not shown). Neithercontrol IgG1 antibody nor 8H9-negative cell lines (Jurkat or HTB119)showed the 90 kD antigen. Following N-glycanase treatment, a single 58kD band was found. O-glycanase had no effect. We interpreted this tomean a protein with heterogeneous glycosylation pattern, withoutdisulfide-linked subunits.

DISCUSSION

We describe a novel 58 kD surface tumor antigen, which is detected bythe monoclonal antibody 8H9. This antigen is expressed on a broadspectrum of human neuroectodermal, mesenchymal and epithelial tumors andappears to be immunohistochemically tumor specific, namely, it isexpressed on cell membranes of tumor cells with no/low membranereactivity noted on normal human tissues. The antigen was present on 88%of neuroectodermal tumors, 96% of mesenchymal tumors and 44% ofepithelial cancers tested. The specific tissue distribution suggests aunique tumor antigen not previously reported.

The expression of the 8H9 antigen on several glial and nonglial braintumors and the complete absence on normal brain tissue is unusual. Thisproperty contrasts with most of the previously described glial tumorantigens with a cell membrane distribution (Table 5).Neuroectodermal-oncofetal antigens e.g. neural cell adhesion moleculesare present to varying degrees on normal adult and fetal tissues (6).Neurohematopoeitic antigens including Thy-1 determinants (24), CD44 (8)and its splice variants (25) are present on normal and neoplastic braintissue as well as hematopoeitic tissues, principally of the lymphoidlineage. Gangliosides, such as G_(D2) and G_(M2), although expressed ontumors of neuroectodermal origin, are also present on normal braintissue (7). The lactotetraose series ganglioside 3′-6″-iso L_(D1) iswidely expressed on brain tumors and on epithelial cancers and germ celltumors as well as on normal brain tissue. (26). TABLE 5 Antigensexpressed on glial tumors Crossreactivity with Antigen Antibody normaltissues Cell Membrane antigens Neurohematopoeitic antigens Thy-1 Ab 390(24) Normal neuronal cells CD44 Multiple Normal endothelium CD44 spliceMultiple (25) Normal neuronal cells variants Cell Adhesion moleculesNCAM ERIC-1 (11), Normal neuronal cells UJ13-A (10) Normal neuronalcells Integrin 3 ONS-M21 (30) Not reactive with normal brainGangliosides G_(D2) 3F8 (35) Normal neuronal cells 3′-6′ iso-LD1 DMAb-22(29) Fetal brain, reactive astrocytes Growth Factor Receptors EGFRvIIIMR1 (9) No normal tissues; breast and lung carcinoma PDGFR- Anti-PDGFR-7(36) Normal neuronal cells Uncharacterized Ependymoma- MabEp-C4 (34) Notreactive with PBL, associated normal brain Glioma- GA-17, GB-4, Notreactive with associated GC-3 (32) normal adult or fetal brain Glioma-6DS1 (33) Not reactive with associated normal adult or fetal brainIntracellular IFAP-300 Anti-IFAP- Not reactive with 300 kDa (37) normalbrain GFAP Multiple Normal neuronal cells Interstitial matrix Tenascin81C6 (13), Normal liver, kidney; not reactive with adult brain BC-2 (14)Not reactive with normal brain GP-240 Mel-14 (38) Melanoma; not reactivewith normal brain Oncofetal BC-1 (39) Adult endometrium; not fibronectinreactive with normal brain

Another remarkable property of the 8H9 antigen is its expression ontumors of diverse lineage: neuroectodermal, mesenchymal and to a lesserdegree epithelial tumors. No monoclonal antibody to date has the bindingspectrum described with 8H9. This broad distribution provides MoAb 8H9the potential of being a “generic” tumor antigen for targeted therapy.Of particular interest is its expression on 28/29 rhabdomyosarcomatumors and the rhabdomyosarcoma cell lines tested by indirectimmunofluorescence. Disseminated and high risk rhabdomyosarcomas have avery poor prognosis with <40% long term survival rate (27). Although theMYOD family of oncofetal proteins are specific to rhabdomyosarcoma, theyare nuclear antigens and therefore unlikely candidates forantibody-based therapy (19). In a preliminary report, cross reactivityof the monoclonal antibody BW575 raised against small cell lungcarcinoma with rhabdomyosarcoma cell lines and 2/2 rhabdomyosarcomasections was described. However, this antibody showed cross-reactivitywith normal tissues (28).

Two further groups of tumors studied were the Ewing's family of tumorsand osteosarcoma. The Ewing's family of tumors can be differentiatedfrom other small blue round cell tumors of childhood by monoclonalantibodies recognizing glycoprotein p30/32 coded by MIC2 oncogene.However, this protein is also expressed on normal tissues and on othertumors, severely limiting its utility in radioimaging and therapy (18).100% (21/21) of Ewing's family tumors tested showed immunoreactivitywith MoAb 8H9. Apart from G_(D2) (15), the osteosarcoma-associatedantigen recognized by the MoAbs TP-1 and TP-3 (17), and the decayaccelerating factor CD55 (16), few tumor-associated antigens have beendefined for osteosarcoma. In our study 28/29 (95%) osteosarcomas testedimmunohistochemically positive with MoAb 8H9. The latter may thereforehave clinical utility in the Ewing's family of tumors and osteosarcomas.

The 8H9 antigen appears to be a novel, previously undescribed antigen.Sensitivity to proteinase suggests that it has a protein component.Conversely, the lack of sensitivity to neuraminidase implies absence ofsialic acid residues, and the lack of sensitivity tophosphatidyl-inositol specific p hospholipase C implies that the 8H9antigen is not GPI anchored. It is unlikely to be related to the neuralcell adhesion molecule family due to its unique distribution andrestriction of expression among normal tissues (6). Of the currentlydescribed antibodies, which bind to glial tumors, four have beenreported to be restricted to tumor tissues. The mutated EGFRvIII wasfound to be expressed on 52% of gliomas tested and crossreacts withbreast and lung carcinomas (29). However, the broad distribution of the8H9 antigen is different from EGFRvIII. Integrin 3, a 140 kDa proteinexpressed on gliomas and medulloblastomas is targeted by the monoclonalantibody ONS-M21 which does not cross react with normal brain (30).However, negative immunoreactivity with neuroblastoma, melanoma andmeningioma has been reported. (31). Similar data on glioma-specificantibodies with no cross reactivity with normal brain has beenpublished. However, they do not react with other neuroectodermal ormesenchymal tumors and data regarding reactivity with other tissues isunavailable (32). A 38 kDa antigen has been targeted on glioblastomacells by the antibody 6DS1. No crossreactivity with human brain has beenreported. Data regarding reactivity with other human tissues is unknown,although a high accumulation of the radiolabeled antibody in mousekidney has been reported. (33). An ependymoma-specific protein antigenof 81 kDa, recognized by monoclonal antibodies which do not crossreactwith normal glial cells, has also been described. These antibodies donot react with other glial tumors such as glioblastoma andcrossreactivity with other tumor tissue is not known (34).

The homogeneous expression of the 8H9 antigen on cell membrane makes itan attractive candidate for targeted immunotherapy. Furthermore, thepersistence of the 8H9 antigen on NMB7 cells after binding to the MoAbsuggests that the antigen is not easily immunomodulated. In order toexplore its potential for radioimaging we used ^(99m)Tc conjugated 8H9to image neuroblastoma xenografts in athymic nude mice. This revealedselective uptake in the xenografts apart from moderate uptake in theliver, % ID/gm being 50% of that achieved with the anti-G_(D2)monoclonal antibody 3F8 (data not shown). The hydrazino-derivative of8H9, therefore, retains the immunoreactive properties of the unmodifiedantibody, and may be useful for radioimaging of tumors. We have alsodemonstrated selective radioimmunolocalization of rhabdomyosarcomaxenografts in athymic mice with no significant uptake in normal tissuesusing ¹²⁵I-labeled 8H9 (data not shown).

In summary, the monoclonal antibody 8H9 recognizes a unique 58 kDtumor-specific antigen with broad distribution across a spectrum oftumors of varying lineage: neuroectodermal, mesenchymal and epithelial,with restricted expression in normal tissues. 8H9 may have clinicalutility in the targeted therapy of these human solid tumors in vitro orin vivo. Further biochemical characterization of the 8H9 antigen iswarranted and may be of interest in delineating a possible role in theoncogenic process.

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Recent clinical trials have shown promising potentials of monoclonalantibodies (MoAbs) in the treatment of cancer: anti-CD20 (lymphoma),anti-HER2 (breast cancer), anti-tenascin (brain tumors), anti-CD33(leukemia), and anti-TAG-72 (colon cancer). In pediatric oncology,tumor-targeting agents are even more relevant since minimal residualdisease (MRD) is often the obstacle to cure, and late effects ofnon-specific therapy are significant. Despite high-intensity combinationtherapy, most metastatic solid tumors (Ewing's sarcoma [ES], primitiveneuroectodermal tumor [PNET], osteosarcoma [OS], desmoplastic smallround cell tumor [DSRT], rhabdomyosarcoma [RMS], and brain tumors)remain incurable. Using metastatic neuroblastoma (NB) for proof ofprinciple, our laboratory integrated the murine IgG3 anti-gangliosideGD₂ MoAb 3F8 into multi-modality therapy. 3F8 has demonstrated highselectivity and sensitivity in radioimmunodetection of metastatictumors, and appears to be a safe and effective method of eliminatingMRD, achieving a >50% progression-free survival (PFS). For mostpediatric solid tumor therapeutic MoAbs do not exist. Known tumorsurface antigens are often restricted to a specific tumor type,heterogeneous in its expression, or found in normal blood cells ororgans. We recently described a MoAb 8H9 which recognizes a novel cellsurface antigen in a wide spectrum of pediatric tumors, with nocrossreactivity with blood, marrow, brain and normal organs, and minimalreactivity with hepatocyte cytoplasm. ¹³¹I or ^(99m)Tc-labeled 8H9 caneffectively image NB and RMS xenografts in SCID mice. Antigen expressionwas generally homogeneous within tumors, and did not modulate on MoAbbinding. We propose to test the targeting potential of ¹³¹I-8H9 in apilot imaging study. Pediatric/adolescent patients with NB, RMS, ES,PNET, OS, DSRT and brain tumors are subjects of our investigation. Wehave two specific aims:

-   -   #1: To measure the level of agreement between conventional        imaging modality (CT, MRI, and nuclear scans) and antibody 8H9        imaging in known and occult sites of disease. Sensitivity        analysis of 8H9 for each disease type will be conducted.    -   #2: To calculate the absorbed dose delivered by ¹³¹I-8H9 to        tumor relative to normal organs.

BACKGROUND AND SIGNIFICANCE

MoAb selective for tumors have therapeutic potential^(1,2) Theintroduction of hybridoma technology by Kohler and Milstein in 1975³ andthe advances in molecular biologic techniques have greatly expanded thepotential of MoAb in human cancers. Optimal targeting of MoAb demandshigh tumor antigen density with homogeneous expression, lack of antigenmodulation on tumor cell surface, adequate vascularity of tumor to allowdeep penetration, minimal toxicity on normal tissues, lowreticulo-endothelial system (RES) uptake, noninterference by circulatingfree antigens, and low immunogenicity. In practice, very fewMoAb-antigen-tumor model systems have fulfilled these stringentcriteria. Recent clinical trials have shown promising potentials ofMoAbs. Anti-CEA antibody in colorectal cancer.⁴, anti-CD20 antibodies inlymphoma,⁵ anti-HER2 antibodies in breast cancer,⁶ anti-tenascinantibodies in glial brain tumors,⁷ MoAb M195 against CD33 in acuteleukemia⁸ and anti-TAG-72 antibodies in colon cancer⁹ have demonstratedefficacy in clinical trials. Our laboratory has developed the MoAb 3F8which targets the ganglioside G_(D2) overexpressed on NB. 3F8 has beenshown to have a high specificity and sensitivity in theradioimmunodetection of minimal residual disease (MRD) in patients withNB,¹⁰ and a significant impact when used as adjuvant therapy.¹¹ ¹³¹I hasbeen a common isotope used both for imaging and therapy purposes.Although not widely available, pure -emitters such as ⁹⁰Y,^(12,13)alpha-emitting particles,^(14,15) such as ²¹¹At, ²¹²Bi and ²¹³Ac haveattractive properties with promising biological effectiveness. Multipleradioisotopes of varying path lengths and half-lives may be needed toenhance radiocurability of both bulk and microscopic diseases. Morerecent developments in immunocytokines (e.g. IL-2, IL-12),¹⁶ bispecificantibodies for pretargeting strategies (e.g. radioisotopes ordrugs),^(17,18) or T-bodies for retargeting immune cells¹⁹⁻²¹ havefurther expanded the potentials of antibody-based immunotherapies.

Brain tumor antigens Examples of tumor antigens expressed on glialtumors include neuroectodermal-oncofetal antigens eg. neural celladhesion molecules (NCAM),²² gangliosides (GD2, GM2, 3′-6″-isoLD1)^(23,24) and neurohematopoeitic antigens (Thy-1, CD44 and splicevariants).²⁵⁻²⁷ All of these antigens are present to varying degrees onnormal adult and fetal tissues, and for some hematopoeitic tissues aswell. Notwithstanding the universal expression of NCAM by neuronalcells, anti-NCAM MoAb UJ13A was shown to accumulate in gliomas by virtueof disruption of blood brain barrier locally²⁸ and another MoAb ERIC-1showed clinical benefit in resected glioma cavities.²⁹ Integrin-3, a 140kDa protein expressed on gliomas and medulloblastomas and not in normalbrain, is a potential target (MoAb ONS-M21)³⁰, but it is poorlyexpressed among other tumor types.³¹ The extracellular matrix proteintenascin is expressed in 50-95% of gliomas as well as on mesenchymaltumors, carcinomas, normal human glial, liver and kidney cells.³²Anti-tenascin monoclonal antibodies 81C6,⁷ BC-2 and BC4³³ administereddirectly into tumor-cavities have shown efficacy in patients withmalignant gliomas. More recent investigations have focused on growthfactor receptors in particular type III mutant epidermal growth factorreceptor (EGFRvIII) expressed on 52% of gliomas³⁴ as well as breast andlung carcinomas.³⁵ Given the relationship of these mutated receptors totheir malignant potential, they may be ideal targets for MoAb. Althoughother glioma-specific antibodies with no cross reactivity with normalbrain have been described (e.g. 6DS1, MabEp-C4),^(36,38) they havelimited reactivity with other neuroectodermal or mesenchymal tumors, anddata regarding cross-reactivity with normal tissues are not available.To date, with the exception of EGFRvIII, the glial tumor antigensdescribed are either found on normal brain and/or normal tissues,restricted to specific tumor types, or found in intracellularcompartments/extracellular matrix which can limit their clinical utilityfor targeting to single cells or spheroids.

Sarcoma antigens Optimal tumor antigens, similarly, have not beendefined for the large family of sarcomas. Although the MyoD family ofoncofetal proteins are specific to rhabdomyosarcoma, they are localizedto the nucleus and therefore do not offer targets for antibody-basedtherapy.³⁹ The ES family of tumors can be differentiated from othersmall blue round cell tumors of childhood by MoAbs recognizingglycoprotein p30/32 coded by the MIC2 oncogene. However, this protein isexpressed on normal tissues (e.g. T-cells)⁴⁰ greatly limiting theutility of MoAb in marrow purging, radioimaging or radiotherapy.⁴¹Membrane targets on OS include GD2,⁴² glycoprotein p72,⁴³ CD55⁴⁴erB2/neu⁴⁵ and the antigen recognized by the MoAb TP-3.⁴⁶ CD55 isdecay-accelerating factor, a ubiquitous protein on blood cells and mosttissues to prevent complement activation. Clearly MoAb directed at CD55would have significant limitations for in vivo targeting. The degree oftumor heterogeneity (e.g. erbB2 in OS) may also limit the efficacy ofMoAb-targeted approach. The presence of G_(D2) on pain fibers causessignificant pain side effects in clinical trials. Nevertheless, thisside effect is self-limited and this cross-reactivity did not interferewith the biodistribution and clinical efficacy of specific MoAb (seepreliminary results). Nevertheless, GD2 is generally low or absent inRMS, ES, PNET, and many soft-tissue sarcomas. In addition, the presenceof G_(D2) in central neurons can limit its application in tumors arisingor metastatic to the brain. Our laboratory has generated a novel MoAb8H9 by hyperimmunizing female BALB/c mice with human NB.⁴⁷ 8H9recognizes a unique surface antigen homogeneously expressed on cellmembranes of a broad spectrum of tumors of neuroectodermal, mesenchymaland epithelial origin, with restricted distribution on normal tissues(see preliminary results).⁴⁸

The availability of an antibody with broad specificity for pediatrictumors will facilitate several lines of clinical investigations. Invitro, such antibodies will be extremely useful for (1) detecting lymphnode or marrow metastasis,⁴⁹ (2) enrichment/isolation of circulatingtumor cells for RT-PCR detection strategies,⁵⁰ (3) purging of bonemarrow before autologous bone marrow transplantation,⁵¹ (4) purging ofex vivo activated T-cells prior to adoptive cell therapy. In vivo itsutility can go beyond its diagnostic capability. When chimerized with ahuman-1Fc tail, it becomes tumoricidal through complement-mediated, andantibody-dependent cell-mediated cytotoxicities.⁵² Through single-chainFv constructs, new fusion proteins can now be delivered to tumor sites(e.g. IL-2, IL-12, toxins, or enzymes). Bivalent scFv and tetravalentscFv can be engineered to improve avidity.⁵³ Bispecific scFv can beconstructed to engage cells and proteins in various targeting strategies(e.g. pretargeting).^(17,18) ScFv can also be used in T-bodies toretarget T-cells, a powerful technique to increase clonal frequency andbypassing the HLA requirement of TCR functions.¹⁹⁻²¹ FurthermorescFv-fasion proteins (e.g. CD28, zeta chain) transduced into T-cells cangreatly enhance their survival following activation.²¹ Even moreimportantly, the ability of such cells to proliferate in contact withtumor cells can further amplify the efficiency of T-cell cytotherapy.

Radioimmunoscintigraphy can test If an antibody-antigen system hastargeting potential. Using radioiodines and technetium we havedemonstrated the utility of the GD2 system for targeting in the lastdecade. This information has been translated into treatment strategiesusing both unlabeled and ¹³¹I labeled antibody 3F8. Dosimetrycalculations have allowed quantitative estimates of therapeutic indexwhen cytotoxic agents are delivered through antibody-based methods.Uptake (peak dose and area under the curve AUC) in specific organsrelative to tumor can be measured. These studies are resource intensiveand to be done well, require laboratory, radiochemistry, nuclearmedicine, medical physics and clinical resource support, as well assubstantial personnel effort. In pediatric patients, issues oftherapeutic index may be even more pressing given the potential of lateeffects of treatment. In addition, despite the potential life-yearssaved for pediatric cancer, orphan drugs are not economically attractivefor most industrial sponsors. These circumstances have made the initialstages of clinical development even more stringent and relatively moredifficult to accomplish.

Patient monitoring and correlative laboratory studies Pharmacokineticstudies are crucial in our understanding of antibody targeting, itstoxicity and its efficacy. Radioimmunoscintigraphy uses the trace labelprinciple and gamma imaging to define the distribution of a specificantibody in various human organs. It provides estimates of antibody (andradiation) dose delivered to blood, marrow and major organs. Thecontinual development of improved software and hardware for calculatingantibody deposits in tissues is critical in implementing these studies(see preliminary results). The quantitative relationship of freecirculating antigens (if present) and biodistribution of MoAb needs tobe defined. The formation of human-anti-mouse antibody (HAMA) responsewill clearly affect the in vivo properties of these antibodies. However,the induction of the idiotype network (see preliminary results) may havepotential benefit in the long run. These parameters need to bemonitored. These in vitro assays will provide important information inunderstanding and optimization of future use of 8H9 and other MoAb inthe context of chemo-radiotherapy for a broad category of recalcitranttumors in children, adolescents and young adults.

Memorial Sloan-Kettering Cancer Center (MSKCC) is devoted to theresearch and clinical care of cancer patients. The Center has anextensive patient referral base, particularly within the tri-state area.The center has an established commitment and past record in the use ofmonoclonal antibodies in the diagnosis and therapy of human cancers,including melanoma, colon cancer, and leukemias. Over the past 4 yearswe have an annual accrual of around 45 new NB, 27 OSs, 58 brain tumors,23 Ewing's/PNET, 18 retinoblastoma, 12 rhabdomyosarcomas, 16 sarcomasand 7 DSRT at MSKCC. We are confident that we can accrue 60 patientswithin the next 2 years. In this past decade, we tested the utility ofMoAb in the curative treatment of a lethal tumor (metastatic stage 4 NBin children). For this orphan disease, the lack ofcorporate/pharmaceutical sponsor has made our progress slow anddifficult. Nevertheless, we made the following observations. (1) MoAbcan extend the progression-free period in a cancer that was uniformlylethal two decades ago. (2) It is feasible to integrate MoAb intostandard chemo-radiotherapy strategies, in order to derive maximalbenefit from all available modalities. (3) Immune based therapies can beadministered safely in the outpatient setting, thus reducing expensivein-patient costs and maximizing time in the home environment (4) MoAbcan induce idiotype network, a potential endpoint that underlies thebiology in maintaining continual clinical remission. (5) GD2 is a usefulmarker of MRD, and specific MoAbs are highly efficacious in monitoringand purging of tumor cells. (6) Novel bioengineering strategies havebeen developed for the GD2-3F8 antigen-antibody system which aredirectly applicable to other MoAbs (single chain Fv,⁵⁴ and T-bodies⁵⁵).During this period, >240 patients have been treated at Memorial Hospitalwith the antibody 3F8. A total of >3500 doses of unlabeled 3F8 have beengiven, 250 injections of ¹³¹I-3F8 for imaging, and 372 injections of¹³¹I-3F8 for therapy. Although there were side effects, there were nolethal sequella during or immediately after antibody administration. 3F8treatment is now routinely done in the outpatient clinic. Extendingthese findings to a second antigen-antibody system, especially one thatwill target to a broader spectrum of pediatric solid tumors is apriority. The murine IgG1 antibody 8H9 has obvious potential inmonitoring and purging of MRD, radioimmunoscintigraphy, andradioimmunotherapy (both intravenous or compartmental). If our proposedstudy produces favorable results, i.e. selective tumor uptake at optimalAUC ratios (Tumor: tissues/organs), radioimmunotherapy can be exploredfor some of these solid tumors. More importantly, further development ofthe antibody would involve a major effort in humanizing and furthergenetic engineering to improve effector functions.

PRELIMINARY RESULTS

G_(D2)-specific MoAb-based targeted therapy: a curative approach to apediatric solid tumor: metastatic NB Improved understanding of thebiology of NB has reshaped our clinical approach to this cancer.Non-infant stage 4 NB remains a therapeutic challenge despite fourdecades of combination chemotherapy. Similar to many cancers, MRD statecan be achieved in patients with NB after intensive inductiontherapy.^(56,57) Unfortunately, the transition from MRD to cure was aformidable hurdle.⁵⁶ Targeted immunotherapy besides being more specificand less toxic, may supplement what chemoradiotherapy has notaccomplished.^(58,59) Disialoganglioside G_(D2) is a tumor antigen wellsuited for targeting therapy because (1) it is expressed at a highdensity in human NB, is restricted to neuroectodermal tissues and isgenetically stable, unlike other tumor antigens such as immunoglobulinidiotypes;⁶⁰ (2) although it circulates in patients' serum, it does notinterfere with the biodistribution of specific antibody (e.g. 3F8),allowing excellent tumor localization of NBs in patients;¹⁰ (3) it isnot modulated from cell surface upon binding to antibodies; (4) it isexpressed homogeneously in human NB, with little heterogeneity withintumors and among patients. Several antibodies against G_(D2) antigen hasbeen described (3F8, 14.G2a, 14.18).^(47,61) In vitro they can targetlymphocytes,^(62,63) granulocytes,^(52,64,65) complement,^(66,67)activated monocytes/macrophages,^(68,69), IL2,⁷⁰,isotopes,^(10,59,71,72) toxins,^(73,74) and superantigen.⁷⁵ Phase I andphase II studies have shown only modest efficacy,⁷⁶⁻⁸⁴ marrow diseasemore likely to respond than bulky tumors.⁸⁵ The major side effectsincluded pain, allergic reactions and neuropathy.^(78,85) With longfollowup, the role of these anti-C_(D2) antibodies at the time of MRDappears promising.

Radiolabeled anti-G_(D2) antibody 3F8 3F8 is a murine IgG₃ MoAb directedat the ganglioside G_(D2) expressed on human NB cells. In preclinicalstudies ¹³¹I-3F8 targeted to human NB xenografts with exceptionally high% ID/gm. Intravenous ¹³¹I-labeled IgG₃ MoAb 3F8 produced a substantialdose dependent shrinkage of established NB in preclinical studies. Dosecalculations suggested that tumors that received more than 4,200 radswere completely ablated. Marrow suppression was the dose limitingtoxicity. In patient studies, it is not trapped nonspecifically by thereticuloendothelial system and penetrates NBs well (0.04 to 0.11%injected dose/gm).^(10,86) Because of the intact blood brain barrier,¹³¹I-3F8 does not normally localize to brain, spinal cord or penetratethe surrounding CSF.^(10,59)

¹³¹I-3F8 is more sensitive than conventional modalities, includingmetaiodobenzylguanidine (MIBG) in detecting NB in patients. Thebiodistribution of ¹³¹I-3F8 was studied in 42 patients (2 mCi perpatient) with NB.¹⁰ Comparison was made with ¹³¹I-MIBG, ^(99m)Tc-MDP(technetium-labeled methylene diphosphonate) bone scan, as well as CT orMRI. ¹³¹I-3F8 detected more abnormal sites (283) than ¹³¹I-MIBG (138) or^(99m)Tc-MDP (69), especially in patients with extensive disease. In 20patients with soft tissue tumors demonstrated by CT/MRI, ¹³¹I-3F8detected the disease in 18 of them. Upon surgical resection, the two¹³¹I-3F8-imaging-negative tumors revealed ganglioneuroma, one showingmicroscopic foci of NB. In contrast, ¹³¹I-3F8-imaging-positive tumorswere all confirmed as NBs. In 26 patients with evidence of marrowdisease by antibody scans, 14/26 had confirmation by iliac crest marrowaspirate/biopsy examinations. Agreement between the measured tissueradioactivity and the estimates based on planar scintigraphy validatedthe initial dosimetry calculations. The tumor uptake in patients with NBwas 0.08%-0.1% ID/gm. The calculated radiation dose was 36 rads/mCidelivered to NB and 3-5 rad/mCi to blood.

¹³¹I-3F8 differentiated Gliomas from normal brain tissues.^(87,88) In 12patients with brain tumors, 3F8 immunoscintigraphy was compared with^(99m)Tc-glucoheptonate/DTPA planar imaging, Thallium 201 single photonemission tomography (SPECT), and ¹⁸FDG positron emission tomography(PET). 10/11l malignant gliomas and 1/1 metastatic melanoma showedantibody localization No nonspecific uptake in normal brain or CSF wasdetected. Average plasma and total body clearance were 20 h and 47 h,respectively. Antibody localization was measured on surgical specimensand time activity curves were calculated based on modified conjugateviews or PET. Radioactivity uptake in high grade glioma peaked at 39 h,which then decayed with a half-life of 62 h. Tumor uptake at time ofsurgery averaged 3.5% ID/kg and highest activity by conjugate viewmethod averaged 9.2% ID/kg (3.5 to 17.8).

Both primary and metastatic Small Cell Lung Cancer were detected by¹³¹I-3F8⁸⁹ 10 Patients with SCLC were imaged with ¹³¹I-3F8. Fivepatients previously treated with chemoradiotherapy were imaged with 2mCi at the time of recurrence, while 5 patients were studied with 10mCi/1.73 m² at the time of diagnosis. No significant toxicities wereseen. All 10/10 tumors showed localization. Precision of localizationwas confined by comparing SPECT and CT in the 5 patients injected withthe 10 mCi dose. Average half-lives for plasma and total body clearancewere 15 h and 58 h, respectively. The tumor to non-tumor ratios appearedfavorable based on the % ID/gm (see below). TABLE 3 % ID/kg after¹³¹I-3F8 Injection: Day Small Spinal Large Tumor sampled Heart BowelSpleen Liver Cord Bowel Blood Muscle Kidney NB 4 1.7 1.7 1.7 2 2.2 2.43.1 3.1 3.1 SCLC 6 0.4 0.4 0.9 0.4 — — — 0.2 0.9 liver Tumor Lung BoneOvaries Adrenal Bladder Stomach Tumor mets NB 3.6 4 4 5.7 6.7 6.7 40 —SCLC 0.5 — — 1.6 0.5 — 2 15

Myeloablative doses of ¹³¹I-3F8 are effective for NB with minimalextramedullary toxicities. Based on the tracer dose dosimetry, theabsorbed doses to liver, spleen, red marrow, lung, total body and tumorwere 537, 574, 445, 454, 499 and 4926 rads, respectively. The averagerad/mCi were 2.3, 2.5, 2, 2, 1.9, and 13.7, respectively. The chemicaltoxicities of the antibody 3F8 have been studied in phase I^(76,77) andphase II studies.^(11,90) Acute toxicities included pain, urticaria,fever and hypotension which were self-limited. The radiologicaltoxicities of ¹³¹I-3F8 were recently defined in a phase I doseescalation study. (6, 8, 12, 16, 20, 24, and 28 mCi/kg).⁹¹ Among 10patients (pts) with progressive disease evaluable for response, 2cleared the marrow and 2 had partial responses of soft tissue tumors.Average tumor dose was 150 rad/mCi/kg. Acute toxicities of ¹³¹I-3F8treatment included pain (20/24) during the infusion, fever (20/24) andmild diarrhea. All pts developed grade 4 myelosuppression. 22/24 ptswere rescued with cryopreserved autologous bone marrow; one patientreceived GM-CSF; one died of progressive disease before marrowreinfusion. Hypothyroidism developed in despite thyroid blockade withoral SSKI plus synthroid or cytomel. In the subsequent phase II study(N7, IRB94-11, FIG. 1), ¹³¹I-3F8 was used to consolidate >50 patients atthe end of induction chemotherapy for their stage 4 NB diagnosed after 1year of age. Except for hypothyroidism, there were no late effects of¹³¹I-3F8 treatment.

¹²⁴I-3F8 PET imaging was first successfully applied to NB⁹² PositronEmission Tomography (PET) can offer advantages over planar or singlephoton emission computed tomography (SPECT) imaging in the quantitationof spatial radioactivity distribution over time. ¹²⁴I is a positronemitter with a 4-day half-life. We have studied the quantitativecapability of PET imaging with ¹²⁴I,⁹³ and have used it for scanning of¹²⁴I-labeled antibodies in animals and humans.^(92,94,95) Using a brainPET scanner (PC4600, Cyclotron Corp.), with a relatively low resolution(FWHM=1.2 cm), we demonstrated that quantitation of ¹²⁴I is possible(range examined was 0.4 to 4 uCi/ml). Studies using ¹²⁴I in a rat tumor(4 gram) measured with this PET scanner were within 8% of the ex-vivomeasurement. Subsequently, two patients were studied on this scannerusing ¹²⁴I-labeled 3F8 antibody.^(88,92) A 3-compartment model was usedto study the kinetics of the antibody to provide an estimate of thebinding potential of 3F8 antibody for glioma. These quantitative studieshave also allowed us to estimate the radiation dose to the tumor cellnucleus from low energy Auger electrons.⁸⁸ More accurate quantitation of¹²⁴I is now possible with the GE body PET scanner with even higherresolution.

¹³¹I-3F8 therapy of leptomeningeal cancer⁹⁶ While overt meningealdisease is rapidly fatal, microscopic deposits in the cranio-spinal axiswill spread even if the primary tumor is eradicated. The potentials ofantibody-derived ligands for the diagnosis and therapy of LM cancer havenot been fully explored. G_(D2) is present on a broad spectrum of humantumors including medulloblastomas, high-grade astrocytomas, PNET,central NBs, small cell lung cancer, melanoma, sarcomas,leukemia/lymphomas and peripheral NBs, many of which have LM spread.Clinical trials using intravenous injections of anti-G_(D2) MoAb 3F8have not encountered long-term neurotoxicity in patients followed for upto 13 years. Pharmacokinetic studies in rats showed that at least 50% ofintraventricular ¹³¹I-3F8 was eliminated by bulk flow. When humanmelanoma leptomeningeal xenografts were present, CSF radioactivity wasretained and AUC (area under curve) increased by 1.5 fold. AUC ratios oftumor to CSF, tumor to brain and tumor to blood were 14, 86, and 64,respectively. These ratios improved to 15, 209 and 97, respectively, ifthe rats were pretreated with diuretics. Direct intraventricularadministration of 30 mCi of ¹³¹I-3F8 in cynomolgus monkeys did notinduce clinical or histological toxicity. Since G_(D2) tissuedistribution (CNS and peripheral) in the cynomolgus monkey is identicalto that of human, the high radiation dose of IT ¹³¹I-3F8 (up to 82 Gy)to CSF in contrast to blood (<2 Gy) may translate into a meaningfultreatment approach. Moreover, serum antibody against the MoAb (AMA) was14-22 fold higher than in the CSF, thereby accelerating blood clearance(reducing blood radiation dose) without affecting CSF pharmacolinetics.

Intra-CSF ¹³¹I-3F8 imaged G_(D2)-positive LM cancers successfully inpatients. The pilot study included 5 patients who had a histologicallyconfirmed diagnosis of a malignancy expressing G_(D2) with LM diseaserefractory to conventional therapies or for which no conventionaltherapy exists. Ommaya catheter placement, patency and CSF flow wasevaluated by ¹¹¹In DTPA studies. Five patients (ages 1-61 years) withleptomeningeal or intraventricular melanoma, ependymoma, rhabdoid tumor(n=2) and retinoblastoma were evaluated. Active disease was identifiedby MR scans in 4 of 5 pts, and by positive CSF cytology in 2. Doses of0.7-1.9 mCi of ¹³¹I-3F8 were injected by Onmuaya catheter. Acute sideeffects included fever (n=2), and headache (n=2) both treated withtylenol, and one episode of vomiting (n=1). One pt had an elevatedopening CSF pressure that remained increased for 36-48 hourspost-injection. There was no appreciable change in WBC, platelet counts,liver or kidney functions tests or CSF cell counts in all 5 patients.

The CSF radioactivity biological half-life, distribution ofradioactivity in the craniospinal axis, and dosimetry at plaques ofdisease and surrounding normal tissues were determined by ¹³¹I-3F8Single Photon Emission Tomography (SPECT). Peak CSF values were achievedgenerally within the first hour of injection. The CSF biologicalhalf-life was 3-12.9 hours, and was in close agreement with the SPECT(7.2-13.1 hours). Estimated dose to the CSF was 14.9-56 cGy/mCi by CSFsamples and 15-31 cGy/mCi by SPECT analysis. Focal areas of tumor uptakewere 27-123 cGy/mCi by SPECT estimates. The radiation dose to the bloodwas 0.9-1.9 cGy/mCi based on blood radioactivity measurements.Post-injection ¹³¹I-3F8 SPECT scans showed distribution throughout thesubarachnoid space along the spinal cord down to the level of the caudaequina by 4 hours, and progressively over the convexity by 24 hours inall patients. Focal ¹³¹I-3F8 uptake was demonstrated in the ventricles,spine and midbrain in 4 patients, corresponding to disease seen on MR.In the one patient who had no MR evidence of disease, ¹³¹I-3F8 clearancewas most rapid (3 hours), with no focal accumulation observed on SPECT.Four patients with focal ¹³¹I-3F8 uptake received 10 mCi of ¹³¹I-3F8through the Ommaya reservoir as part of a treatment protocol in a phaseI toxicity study. Except for grade 2 toxicities (fever, headache, nauseaand vomiting, increase in intracranial pressure) and a breakthroughseizure, there were no adverse side effects during their initialtreatment One patient had a radiographic and clinical response. Onrepeat treatment 2 months later, with the same dose, a rapid rise ofintracranial pressure necessitated a shunt placement.

Although all 4 treated patients progressed, 3 are still alive (2+, 3+and 9+ months from treatment).

Adjuvant anti-G_(D2) antibody 3F8 3F8 (without radioisotope) has alsobeen tested in phase I and phase II studies.^(58,76,77) Responses ofmetastatic NB in the bone marrow were seen. Another mouse antibody14.G2a and its chimeric form 14.18 have also induced marrow remissionsin patients with NB.⁸³ Acute self limited toxicities of 3F8 treatmentwere pain, fever, urticaria, hypertension, anaphylactoid reactions, aswell as decreases in blood counts and serum complement levels, and inrare patients self-limited neuropathy.^(71,97-99)

Anti-GD2 antibody treatment of MRD in stage 4 NB diagnosed at more thanone year of age.¹¹ Thirty-four patients (pts) were treated with 3F8 atthe end of chemotherapy. Most had either bone marrow (31 pts) or distantbony metastases (29 pts). Thirteen pts were treated at second orsubsequent remission (group I), and 12 pts in this group had a historyof progressive/persistent disease after ABMT; 21 pts (all on N6protocol) were treated in first remission following inductionchemotherapy (group II). At the time of 3F8 treatment, all 34 patientshad stable or minimal NB. Twenty-three patients were in CR, 8 in VGPR, 1PR and 2 with histological evidence of marrow disease. Since microscopicoccult NB could escape detection by conventional radiographic studies,three additional sensitive methods were used to document disease priorto 3F8 treatment. They were 131I-3F8 immunoscintigraphy, marrowimmunocytology, and molecular detection of marrow GAGE by RT-PCR.Fourteen of 34 patients were 131I-3F8 scan-positive prior to 3F8treatment. Nine had residual disease in their marrow by immunocytologyand 12 had evidence of marrow disease by RT-PCR. A total of 25/34patients were positive for disease by at least one of these threemethods. Thirteen patients are progression-free (40 to 148+ months fromthe initiation of 3F8 treatment); one other patient is alive withdisease 61+ months after 3F8 treatment Both group I and group IIpatients achieved long-term progression-free probabilities of 38%. Amongthe 20 patients whose disease progressed after 3F8, 3 in group II hadisolated relapse in the CNS, a sanctuary site where antibody 3F8 couldnot penetrate.86 Although the majority of patients were in CR/V/GPR byconventional criteria right before 3F8 treatment, 74% had evidence ofdisease by the more sensitive methods (immunoscintigraphy with 131I-3F8,bone marrow immunocytology and RT-PCR). When these tests were repeatedsubsequent to 3F8 treatment, 6/9 patients with positive immunocytologyreverted to undetectable. Among the 12 GAGE-positive patients, 7 becamenegative for GAGE expression. Six patients had post-3F8 treatment131I-3F8 scans and all 6 showed resolution or improvement

Human anti-mouse antibody response (HAMA) and patient outcome: Threepatterns of HAMA response were identified. In pattern I, HAMA was notdetectable during the 4-6 month followup period after first cycle of3F8, 42% had no HAMA response even after receiving 2-4 cycles of 3F8over a 4-25 month period. In pattern II, HAMA was detected but rapidlybecame negative during the 4-6 month followup period. In pattern III,HAMA titer was high (>5000 U/ml) and persistent during the 4-6 monthfollowup period. When patients developed HAMA (>1000 U/ml) during atreatment cycle, pain side effects disappeared. In the absence of HAMA(pattern I) or when HAMA became negative (pattern II), patients receivedrepeat 3F8 treatments. In the presence of HAMA, subsequent 3F8treatments had to be delayed. Thus, patients in group III did not getrepeat 3F8 treatment during the first 4-6 months, and had fewertotal-cycles and fewer total-days of 3F8 treatment, while pattern I andII patients were comparable. Kaplan Meier analysis showed a survivaladvantage for those with pattern II HAMA response, i.e. a lowself-limiting HAMA response (73% for pattern II versus 33% for patternI, and 18% for pattern III). The probability of survival among patientswith pattern II was significantly better than the pattern I and IIIpatients combined (p<0.05). For patients progression-free for at least12 months after the last cycle of chemotherapy, those receiving four 3F8cycles had a PFS probability double those receiving less than 4 cycles(p=0.08). When patients with pattern II HAMA response and/or four cyclesof 3F8 were considered as a group (FIG. 1), their survival wassignificantly better than the other 20 pts (p<0.001). We interpret thesefindings to mean a threshold (four 3F8 cycles, each 10-day cycles) plusa pattern II HAMA response may be necessary to maintain permanent tumorcontrol.

Idiotype network is a possible mechanism for long term PFS. Since theHAMA response was primarily anti-idiotypic (Ab2), we postulate that thesubsequent induction of an idiotype network which includedanti-anti-idiotypic (Ab3) and anti-G_(D2) (Ab3′) responses may beresponsible for tumor control in patients. Their serum HAMA, Ab3, andAb3′ titers prior to, at 6, and at 14 months after antibody treatmentwere measured by ELISA. Long term PFS and survival correlatedsignificantly with Ab3′ (anti-G_(D2)) response at 6 months, and with Ab3response at 6 and 14 months. Non-idiotype antibody responses(anti-mouse-IgG3 or anti-tumor nuclear HUD antigen) had no apparentimpact on PFS or survival. It appears that the successful induction ofan idiotype network in patients maybe responsible for long term tumorcontrol and prevention of late relapse among N6 and N7 patients (FIG.2). Even among patients treated on N5 (with ABMT, FIG. 2), all of thesurvivors of bony and marrow metastases have had imaging studies with3F8 and had detectable idiotype network by ELISA¹⁰⁰; similarly no laterelapses were seen. While N5 and N6 groups had no relapses after ˜3years from diagnosis or ˜2 years from 3F8 therapy (including secondremission group), among N7 patients, the relapse curve has leveled offeven earlier, around 2 years from diagnosis.

Integration of 3F8 treatment into multi-modality therapy: N5, N6 and N7for stage 4 NB>1 year of age: From 1987 to 1999, N5, N6 and N7 protocolswere designed sequentially to test the clinical importance of doseintensity, 3F8, and ¹³¹I-3F8 in consecutive patients with newlydiagnosed stage 4 NB. Most of them had very high-risk clinical andbiologic markers, almost all were diploid/tetraploid and of unfavorablehistopathology. Except for ¹³¹I-3F8 and autologous marrow transplant(ABMT), chemotherapy and 3F8 are routine outpatient procedures.Evaluations at sequential endpoints compared favorably with predictions:primary tumor resectability, overall response, and progression-freesurvival (PFS). There were no late relapses after 3.5 years fromdiagnosis. For N6 (all survivors past 5 years) 40% are progression-free;for N7, PFS is projected at 55% (p=0.02 when compared to N5). Causes ofdeath included disease progression, secondary leukemia, and isolated CNSrelapse. Although toxicities included hearing loss and hypothyroidismwhich required correction, a curative strategy for stage 4 NB appearedto be within reach.

Neuroblastoma, 3F8 and GD2 provided us with the proof of principle thatMoAb may have potential in the permanent eradication of MRD in thecurative treatment of solid tumors in the younger population. Both RITand idiotype-network induction are possible with murine MoAb. Wetherefore undertook an extensive screening of MoAbs to identifycandidates with a broad reactivity with pediatric/adolescent solidtumors, that may have similar targeting potential as the antibody 3F8.

Novel antigen for MoAb targeting to solid tumors in children and youngadults Female BALB/c mice were hyperimmunized with human neuroblastomaaccording to previously outlined methods.⁴⁷ Splenic lymphocytes werefused with SP-2/0 mouse myeloma cells line. Clones were selected forspecific binding to neuroblastoma on ELISA. The 8H9 hybridoma secretingan IgG1 monoclonal antibody was s elected for further characterizationafter subcloning.

Normal and tumor tissue reactivity of 8H9 antibody Frozen sections from315 tumors with histologically confirmed diagnoses of cancer wereanalyzed for immunoreactivity with MoAb 8H9. (Tables 5 and 6) 15histologically normal human tissues and 8 normal monkey tissues werealso analyzed ( ). TABLE 5 8H9 Neuroectodermal Tumors No. positive % NB87 84 97 Brain Tumors 1. Glial Tumors Glioblastomas multiforme 17 15 88Mixed Glioma 4 3 — Oligodendroglioma 11 4 36 Astrocytoma 8 6 75Ependymoma 3 2 — 2. Primitive PNET Medulloblastoma 2 2 — 3. MixedNeuronoglial tumor 2 1 — 4. Other Schwannoma 3 3 — Meningioma 2 2 —Neurofibroma 1 1 Melanoma 16 12 75 Ewing's Family of tumors 21 21 100TOTAL 177 156 88

TABLE 6 8H9 Mesenchymal Tumors No. Reactive % Rhabdomyosarcoma 26 25 96Osteosarcoma 26 25 96 Desmoplastic small round cell tumor 34 32 94Malignant fibrous histiocytoma 1 1 — Synovial sarcoma 2 1 —Leiomyosarcoma 4 4 — Undifferentiated sarcoma 2 2 — TOTAL 95 90 95

TABLE 7 CARCINOMAS No. 8H9 Reactive % Breast 12 4 33 Bladder 4 1 —Adrenal 2 1 — Stomach 1 1 — Prostate 2 1 — Colon 2 1 — Lung 1 1 —Endometrium 1 1 — Cervix 1 0 — Renal 1 1 — TOTAL 27 12 44

TABLE 8 Other Tumors No. 8H9 reactive % Hepatoblastoma 4 3 — Wilm'stumor 8 7 — Rhabdoid tumor 3 3 — Paraganglioma 1 1 — TOTAL 16 14 88

Heterogenous, non-specific cytoplasmic staining was noticed in normalhuman pancreas, 5 stomach, liver and adrenal cortex which was diminishedwhen 8H9 F(ab′)2 fragments were used instead of the whole antibody forimmunostaining. None of the other human tissues showed reactivity with8H9. In particular normal human brain tissue sections including frontallobe, spinal cord, pons and cerebellum were completely negative. Normaltissues from cynomolgus monkey also demonstrated similarly restrictedreactivity with nonspecific staining observed in stomach and liver(Table 4). The majority of neuroectodermal and mesenchymal tumors testedshowed positive reactivity with 8H9, epithelial tumors to a lesserextent. 8H9 immunoreactivity was seen in a characteristic, homogenous,cell membrane distribution in 272 of the 315 (86%) tumor samplesexamined. 88% of n euroectodermal tumors, 95% of m esenchymal tumors and44% of epithelial tumors tested positive with 8H9 (Tables 4-8) TABLE 4Tissues Human Cynomolgous Frontal lobe Negative Negative Pons NegativeNegative Spinal cord Negative — Cerebellum Negative Negative LungNegative — Heart Negative Skeletal muscle Negative — Thyroid Negative —Testes Negative — Pancreas cytoplasmic — staining Adrenal cortexcytoplasmic cytoplasmic staining staining Liver cytoplasmic cytoplasmicstaining staining Stomach — Negative Sigmoid colon Negative — BoneMarrow Negative — Kidney Negative Negative

Indirect immunofluorescence 8H9 immunoreactivity in 34 NB, melanoma,RMS, small cell lung cancer, OS, glioblastoma, leukemia, breast cancerand colon cancer cell lines was tested using indirectimmunofluorescence. Moderate to strong cell membrane reactivity with 8H9was detected in 16/16 NB, 2/2 melanoma, 2/2 RMS, 1/1 glioblastomamultiforme, 3/3 breast cancer, and 1/1 colon cancer, 2 of 3Ewing's/PNET, and 2 of the 3 OS cell lines. The small cell lung cancercell line HTB119 tested negative with 8H9 as did Jurkat T -ALL cell lineand EBV transformed lymphoblastoid cells. Normal human bone marrowmononuclear cells (n=80) and hepatocytes (n=2) had no reactivity with8H9. Hepatocytes were isolated from human cadavers and stained with 8H9.In contrast to anti-cytokeratin 18 and anti-HLA-class-1 antibodies whichreacted strongly with surface antigens, 8H9 staining was equivalent tocontrol antibody.

Antigen modulation 8H9 binding to neuroblastoma line (NMB7),rhabdomysarcoma (HTB82) and OS (U2OS) (measured by indirectimmunofluorescence) did not diminish significantly after 48 hr ofincubation at 37° C. During the same period, binding to HLA (MoAb HB95)diminished by 85% and to GD2 (3F8) by 55%, respectively FIG. 3).

Electron microscopy using gold-labeled antibodies will be moredefinitive in tracking antibody internalization, a process clearlyimportant for immunotoxins to be effective.

Enzyme-sensitivity There was a pronase dose-dependent reduction inreactivity with 8H9 with 75-85% loss of immunofluorescence at a finalPronase concentration of 0.3 mg/ml (FIG. 4). There was no appreciableloss of reactivity with 3F8 (specific for the ganglioside GD2) on NMB7cells. Furthermore, the 8H9 antigen was not sensitive to neuraminidaseor phosphatidyl-inositol specific phospholipase C (data not shown).

Biochemical Characterization of the novel antigen recognized by 8H9Using a nonradioactive cell surface labeling technique, the antigen wasimmunoprecipitated and analyzed on a SDS-PAGE.¹⁰¹ In brief, NB NMB7 orOS U2OS cells were biotinylated using biotin-LC-NHS, lysed, preclearedwith protein-G sepharose, reacted with antibody 8H9. and thenimmunoprecipitated in fresh protein G sepharose. Antigen was thendissociated from the gel and separated by SDS-PAGE. Followingtransblotting onto nitrocellulose membrane, the protein bands weredetected with HRP-strepavidin and visualized by ECL. A band of 90 kDaunder non-denaturing conditions and 96 kDa in the presence of 2 ME wasfound. TABLE 9 % ID/gm TISSUE NB RMS Time 24 h 172 h Tumor 8.3 5.3 Brain0.2 0.1 Heart 2.1 0.8 Lung 0.8 1.4 Kidney 2.3 0.7 Liver 7.5 0.6 Spleen6.7 0.6 Bladder 1.0 1.1 Stomach 0.3 0.5 Sm Intestine 0.3 0.3 LgIntestine 0.4 0.2 Muscle 0.2 0.2 Femur 0.7 0.3 Adrenal 1.0 0.3 Skin 0.20.4 Spine 1.7 0.4 Blood 3.8 3.3

Rat Anti-idiotypic MoAb specific for 8H9 By immunofluorescence theantigen was sensitive to low temperatures. In view of the lability ofthe antigen, we chose to synthesize anti-idiotypic antibodies assurrogate antigen-mimics, in order to allow in vitro monitoring of theantibody immunoreactivity e.g. after iodination of antibody 8H9. LOU/CNrats were immunized with protein-G purified 8H9 precipitated withgoat-anti-mouse Ig, emulsified in CFA. Following in vitro hybridizationto the myelomas SP2/0 or 8653, 3 IgG2a clones (2E9, 1 E12, and 1 F11)were selected for their high binding and specificity. When testedagainst a panel of 23 other myelomas or hybridoma antibodies, nocross-reactivity was found. The anti-idiotypic hybridomas were clonedand antibodies produced by high density miniPERM bioreactor from UnisynTechnologies (Hopkinton, Mass.). The anti-idiotypic antibodies arefurther purified by protein G (Pharmacia) affinity chromatography. Tofurther prove that these anti-idiotypic antibodies are antigen-mimics,we immuno-enrich phagemids and screen scFv on solid phase anti-idiotype,and successfully isolate a number of 8H9-scFv with similar bindingspecificity to tumors as the parent 8H9 (see below).

Tumor localization in xenografted SCID mice SCID mice with NB (NB)xenografts were injected iv with 100 ug ^(99m)Tc labeled 8H9. Bloodclearance was studied by blood cpm at various intervals after injection.Mice were sacrificed at 24 hours and tissue uptake expressed as percentinjected dose per gram (Table 9). Significant uptake in thereticulo-endothelial system in liver and spleen was seen only with^(99m)Tc-8H9; none was evident when ¹³¹I-3F8 was used. There was nosignificant difference between ^(99m)Tc-8H9 and ¹³¹I-8H9biodistribution. When the specific activity of ¹³¹I-8H9 was increasedfrom 5 to >20 mCi/mg, there was no degradation of tumor imaging ordifference in biodistribution. In SCID mice xenografted with RMS (RMS)xenograft following iv injection of 100 uCi of ¹²⁵I-8H9, selective tumoruptake was evident at 4 to 172 hrs after injection, with a blood T½ of0.8 h and T½ of 26 h. Mean tumor/tissue ratios were optimal at 172 h(for lung 4, kidney 7, liver 9, spleen 10, femur 16, muscle 21, brain45). Average tumor/blood ratio were 0.7,1.4 and 1.6, and tumor uptakewas 9.5±3.4, 13.3×1.5, and 5.3±0.9% injected dose per gm at 24, 48 and172 h, respectively. Control IgG1 MoAb antibody 2C9 remained in theblood pool without localization to sc RMS xenografts. Tumor to normaltissue ratio was favorable [range 5-55]for 8H9 (solid bar, FIG. 5) incontrast to control MoAb 2C9.

8H9-ScFv We have synthesized single chain antibody (scFv) from 8H9.Using polymerase chain reaction splicing by overlap extension, variableregions of the heavy (V_(H)) and light chains (V_(L)) of 8H9 were joinedby a polylinker (L) (gly4Ser)₃ and selected by phagemid expression. scFvwas characterized by DNA sequencing, western blots, in vitro ELISA,immunostaining/FACS, and idiotype analysis. Using this scFv as atargeting unit, we are in the process of synthesizing scFv-h 1-CH2-CH3chimeric, scFv-m 3-CH2-CH3 chimeric, and T-bodies for retargetingT-cells.

Cell Populations Using 8H9-Magnetic Bead Immunoselection. ES is a smallround blue cell tumor of childhood characterized by a t(11,22) in mostpatients. Because survival remains suboptimal with standard therapy,many patients receive autologous stem cell transplant and current trialsinvestigating adoptive transfer of autologous T cells in the context ofimmune therapy are underway. However, approximately 50% of patients withadvanced disease have PCR detectable ES in peripheral blood and/or bonemarrow and the administration of autologous cell preparationscontaminated with tumor may contribute to disease relapse. To date,there is no method reported for purging contaminated hematopoietic cellpopulations or bone marrow preparations of ES. Merino et al in thelaboratory of Dr. Mackall at the Pediatric Oncology Branch, NCI,Bethesda, Md., successfully optimized 8H9 for immunomagnetic purging ofES. 8H9 bound to 9/9 of ES cell lines by flow cytometry. Binding toperipheral blood mononuclear T cell and B cell populations, as well asCD34+ cells from bone marrow was negative. Utilizing immunomagneticselection, 8H9 was used to isolate ES cells from contaminated blood cellpopulations. Using real-time quantitative nested PCR with theLightcycler instrument, purging efficiency was monitored by of t(11,22)RT-PCR. Contaminated specimens were reacted with 8H9 and then incubatedwith rat anti-mouse IgG1 magnetic beads. The sample was then run over aMiltenyi Variomax negative selection column. Recovery was approximately70% of the total PBMC. RNA was extracted from 10e7 cells from pre andpost purge cell populations. Real time quantitative PCR was performedwith a level of sensitivity to one tumor cell in 10e5 normal cells. A2-log reduction of tumor cells was achieved at a contamination of onetumor cell in 10 normal PBMC and one tumor cell in 10e3 normal PBMC.Further studies evaluating efficacy in clinical samples are underway.These results demonstrate a potential new approach for purgingcontaminated patient samples to be used in the context of autologousbone marrow transplant and/or immunotherapy trials for ES.

8H9 purging of NB from marrow or blood cells In similar experimentsusing Dynal beads coated with human anti-mouse IgG (Dynal, Lake Success,N.Y.)⁵⁰ EGFP marked NMB7 cells could be quantitatively removed in aone-cycle (either 8H9 or 3F8) or 2-cycle (8H9 followed by 3F8)immunomagnetic strategies (Table 10).

RESEARCH DESIGN AND METHODS

We will test if intravenous injections of iodine-131 labeled murine MoAb8H9 can detect primary and metastatic solid tumors. A total of 60patients will be accrued over a period of 2 years.

#1: To Define the Level of Agreement Between ¹³¹I-8H9 and ConventionalImaging Modalities in the Detection of Primary and Metastatic SolidTumors in Pediatrics.

1.1 Study Design

This is an open-label single arm study of ¹³¹I-8H9, injectedintravenously at 10 mCi/1.73 m2 dose, after which patients will beimaged at approximately day 0 to 1, 2d, 3d and whenever possible 6 to 7dfor dosimetry calculations. Blood samples will also be obtained at least12 times over the ensuing 7 days. Patients are eligible for the protocolprior to their surgical resection or biopsy of known or suspected tumor,or at the time of recurrent tumor. ¹³¹I-8H9 injection plus imaging canbe repeated in each patient up to a total of 3 times, but only if he/shehas no HAMA and no allergy to mouse proteins as evidenced by a negativeskin test

1.2 Patient/Subject Inclusion Criteria

Gender and Minority Inclusion for Research Involving Human Subjects:

Memorial Sloan Kettering Cancer Center has filed form HHS 441 (Re: CivilRights), form HHS 641 (Re: Handicapped individuals), and form 639-A (Re:sex discrimination). In selecting patients for study in the proposedproject, due notice is taken of the NIH Policy concerning inclusion ofwomen and minorities in clinical research populations. The studypopulation will be fully representative of the whole range of patientsseen at Memorial Hospital. No exclusions will be made on the basis ofgender or ethnicity. However, because of the nature of these cancerswhich tend to present in children and young adults, most the humansubjects would be of the younger age group.

Based on a December 1998-November 1999 analysis of the patientpopulation accrued to therapeutic clinical protocols, the racialdistribution of these patients were 16.6% black, Hispanic, or Asian,78.2% white and 5.2% other or unknown. The gender was 55.9% male and44.1% female. For the total patient population diagnosed and treated atMSKCC in 1996, 26% were black, Hispanic, Asian or Native American, 70%white and 6% unknown or not responding. Of these patients, 38% were maleand 62% female.

Participation of children: Children, adolescents and young adults arethe subjects of this clinical trial because of the nature of thesecancers. There is no age limit.

1.3.0 Evaluation During Tireatment/Intervention

1.3.1 After injection of radiolabeled antibody, 1-2 cc of blood inpurple tops (EDTA) will be drawn at time 0, and around 15 min, 30 min, 1h, 2 h, 4 h, 8 h, 18 h, 30 h, 42 h, 66 h, and once on day 6 or 7.Samples should be dated and timed. These samples are for pharmacokineticand for dosimetry studies. Patients with delayed clearance will have onemore imaging done between day 9 to 11. Time Procedure day −10 startdaily oral SSKI, cytomel for thyroid blockade day 0 5 mCi of iodine-131on 0.25 to 0.75 mg of 8H9* blood samples at 0, and approximately 15 min,30 min, 1 h, 2 h, 4 h, 8 h after injection day 0 Gamma camera scan pluswhole body counts day 1 Gamma camera scan plus whole body counts day 1blood samples at approximately 18 h and 30 h day 2 Gamma camera scanplus whole body counts day 2, 3 blood samples at approximately 42 h and66 h day 5 Gamma camera scan plus whole body counts (or 6 or 7) andblood sample day 9(or 10 or 11) if slow clearance Gamma camera scan pluswhole body counts and blood sample day 14 Oral SSKI and cytomeldiscontinued*Premedication with acetaminophen and diphenhydramine.

1.3.2 Patients will undergo gamma imaging days 0, 1, 2 and 5 or 6 or 7after injection.

1.3.3 Blood for HAMA q 1-2 months

1.3.4 Tissue biopsy is recommended for regions of uptake by 8H9 imagingand negative by conventional radiographic techniques.

1.4.0 Biostatistics

To measure the level of agreement between conventional imaging modality(CT, MRI, and nuclear scans) and antibody 8H9 imaging in known andoccult sites of disease. Index lesions will be confirmed either bysurgery or by disease-specific imaging (e.g. MIBG for NB). For eachindividual, the proportion of sites found by 8H9 imaging will be scored.Given that there will be confirmation by surgery or by disease-specificimaging, sensitivity analysis of 8H9 for each disease can be conducted.The probability of agreement or positive predictive value will becalculated. The 95% confidence intervals can be calculated within ±31%for each disease (NB, RMS, ES/PNET, DSRT, brain tumors and othersarcomas). The study will be performed on a total of 60 patients (10with NB, 10 RMS, 10 osteosacrcoma, 10 ES, 10 DSRT and 10 brain tumorsplus other 8H9-positive tumors). Estimates on the level of agreement andthe level of tumor uptake will be computed separately in each diseasegroup. We are not using Kappa statistics for testing the associationbetween ¹²⁴I-3F8 imaging and other imaging modalities (CT, MRI) sinceonly patients with measurable or evaluable tumors will be eligible forthis protocol. In other words, patients with no evidence of disease byconventional studies will be not eligible. Therefore we cannot estimatethe probability of negative 8H9 imaging when conventional imagingstudies are negative, i.e. specificity analysis.

1.5.0 Preparation of ¹³¹I-8H9

8H9 is produced under GMP conditions and packaged in glass vials. ¹³¹Iis purchased from Amersham Inc. 8H9 will be labeled with radioactiveiodine using iodogen T method. The reaction mixture is filtered throughan ion exchange (AG1X8) filter (Biorad) to remove free iodine. Proteinincorporation is measured using TCA precipitation or thin layerchromatography. Immunoreactivity is measured by 2 separate methods (1) asolid phase microtiter radioimmunoassay technique previouslydescribed,¹⁰² and (2) anti-idiotype peak shift method, whereanti-idiotypic antibody 2E9 is added at 50 to 1 molar ratio to ¹³¹I-8H9for 30 minutes on ice with mixing. The percent cpm shifted on HPLC is ameasure of immunoreactivity. Radioiodinated 8H9 has a meantrichloroacetic acid precipitability of >90%, and specific activity of¹³¹I-8H9 averaging 10 mCi per mg protein. Administration of ¹³¹I-8H9 isundertaken within 1-2 hours of iodination to reduce the possibility ofradiolysis. Antibody radiolabeling is carried out in the Central IsotopeLaboratory under the supervision of Dr. Ronald Finn, according to FDAguidelines on radiolabeled biologics for human use.

1.6.0 Infusion of Radiolabeled Antibody Preparation and Monitoring ofPatient Response in Immediate Post-Infusion Period, Including RadiationSafety Aftercare.

All radiolabeled MoAb preparations will be injected into patients by atrained research nurse or physician. Strict observance of appropriateradiation safety guidelines will be undertaken. The procedure will beexplained to the patient thoroughly prior to the infusion by thephysician, and appropriate pre-treatment (eg SSKI drops, perchloracap)checked. The radiolabeled antibody will be transported from theradiolabeling facility to the infusion area loaded into the infusiondelivery system by the physician. The physician and nurse will bepresent throughout the infusion and in the post-infusion period.

The infusion procedure will consist of the radiolabeled antibody beingadministered intravenously either through a peripheral intravenouscatheter or an indwelling central catheter over a 20 minute period. Allpatients will have vital signs monitored prior to and following theradiolabeled antibody infusion. Blood samples for pharmacokineticcalculation will be obtained immediately following the infusion, and atvarious time points thereafter as outlined above. The patient will beseen by a physician daily while hospitalized, and will be available forconsultation (with appropriate radiation safety personnel) with anoncologist or nurse regarding issues relating to the radiolabeledantibody infusion or radiation safety. The patient will also be imagedin the Nuclear Medicine Department over the subsequent two week period,and all imaging procedures performed will be supervised by the physicianto ensure that appropriate studies are obtained.

1.7.0 In vitro Radiolimmunoassay, ELISA, and Immunostaining

Quantitative in vitro assays on biologic fluids collected during thecourse of clinical research studies in individual patients that employradiolabeled antibodies will be carried out. The methods provided willinclude gamma counting of blood samples and HAMA assays. HAMA titer inblood and serum will be correlated with the clearance of ¹³¹I-8H9

1.7.1 General counting procedures Aliquots of whole blood/plasma/serumobtained from patients infused with radiolabeled antibodies will becounted in a gamma counter with standards of known activity fordetermination of sample activity. Tissue samples obtained by biopsy orsurgery will also be counted in a gamma counter for determination of %injected dose/gram tissue. Appropriate quality control procedures willbe observed for counting instruments and tissue specimens.

1.7.2 Quantitation of HAMA by ELISA The presence of HAMA can modify thebiodistribution of ¹³¹I-8H9. Although in naive patients HAMA istypically undetectable, in patients with prior history of exposure tomurine antibodies or to 8H9, the presence of HAMA before and soon after8H9 injection will need to be monitored. In addition, the formation ofHAMA was highly correlated with patient survival in the GD2-3F8 system,we plan to measure the serum antibody titer 6 months and 12 months after8H9 exposure. The ELISA method has been described previously.¹¹ UsingF(ab′)2 fragments derived from the three anti-idiotypic antibodies (2E9,1E12, and 1F11), serum Ab3 will also be monitored as previouslydemonstrated for the GD2-3F8 system.^(103,104)

1.7.3 Quantitation of free circulating antigen Since the biodistibutionof 8H9 will be greatly affected by any soluble antigen, patient serabefore antibody injection will be analyzed for antigenemia using anELISA inhibition assay using a modification of previously describedmethod.¹⁰⁵ Microtiter wells are coated with anti-idiotype MoAb 2E9. Ser.serum dilutions are used to inhibit the binding of biotinylated 8H9,which can be detected by peroxidase-streptavidin. Upon washing, colorreaction is performed at room temperature using hydrogen peroxide assubstrate and o-phenylenediamine (Sigma, St. Louis, Mo.) as chromogen.After stopping the reaction with 30 ul of 5N sulfuric acid, opticaldensity of the wells are then read using MRX microplate reader (Dynex,Chantilly, Va.) and antibody titer calculated in units/ml.

1.7.4 Immunostaining of tumor tissues Tumor tissues will be tested forantigen expression using methods previously described.⁷⁴

Anticipated results and potential pitfalls The injection of ¹³¹I-8H9intravenously or intrathecally into cynomolgus monkeys were welltolerated. Although we do not anticipate any untoward side effects,patients will be closely monitored during the antibody infusion withoxygen, antihistamines, epinephrine, and hydrocortisone at the bed side.After the completion of antibody injection, patients will be observedfor at least 1 hour before discharge from the clinic. Patients withunexpected grade 3-4 (other than urticaria, self-limited bloodpressure/pulse/temperature changes) or any life-threatening toxicitywill be reported immediately to the IRB and FDA. Given the lability ofthe antigen in the cold (whether free or cell-bound), immunoreactivityand soluble tumor antigen will be assayed using the anti-idiotype as theantigen-mimic. The anti-idiotypic antibodies are rat IgG1 MoAb purifiedby acid elution from protein G affinity columns.

They have remained stable despite acid treatment, buffer changes andfreezing and thawing. Soluble antigens can interfere with tumortargeting. In vitro, patient serum did not inhibit binding of 8H9 to itsanti-idiotype. Indirect immunofluorescence of a spectrum of cell linesshowed persistence of antigen and antibody on the cell surface at 37° C.over days. In xenograft biodistribution studies, there was no evidenceof antigen shedding that interferes with tumor imaging. Althoughinterference of 8H9 biodistribution by soluble antigen is unlikely, wewill document the absence by the ELISA inhibition assay. HAMA responsewithin the first two weeks after MoAb injection is rarely observed amongour patient population, partly because of the intensity of thechemotherapy they received. However, some are expected to mount a HAMAresponse when they are imaged a second time. Clearly their HAMA will bemonitored before and after injection in order to interpret thebiodistribution results. Because of this sensitization, these patientsmay not be eligible for subsequent MoAb therapies (as stated in theconsent form). However, we hypothesize that this HAMA response will helpinduce the idiotype network, which may have benefit on patient survival,analogous to our success with the murine 3F8-G_(D2) system we describedin preliminary results and progress report.

Interpretations and implications The ability of 8H9 to detect a broadclass of primary and metastatic solid tumors will be the first step indefining the clinical utility of MoAb 8H9 in vivo. Besides being auseful diagnostic tool, its therapeutic potential will need to beexplored. Clearly the amount of antibody deposited in various organsneed to be taken into account if these antibodies are used to deliverradioisotopes, enzymes or drugs. Chimeric antibodies with improved Fceffector functions and reduced immunogenicity will also be explored.Immunocytokines and T-bodies are also potential steps in futuredevelopment of these agents.

#2: To Estimate the Radiation Dose per mCi of ¹³¹I-8H9 Delivered toTumors and to Normal Organs in Patients.

To obtain data necessary for patient dosimetry, patients will beinjected, intravenously, with ¹³¹l-8H9 according to their surface area,i.e. 10 mCi/1.73 m2. A total of three or four gamma camera images willbe obtained within a 1 to 2 week period following injection. Thefollowing schedule is recommended but may be altered, if necessary: 1-4h after injection (day 0) and then again on days 2, day 3, and day 6 or7. If warranted, due to slow clearance kinetics, imaging on days 9, 10or 11 may also be performed. Using this schedule weekend imaging may beavoided regardless of the weekday injected. Scan types and imagingparameters are listed below:

2.1 Data Collection:

SPOT and SPECT images will be collected over pre-selected “index” tumorlesions, as identified from previously obtained CT or MR images.

2.1.1 Blood collection Blood samples will be collected as follows: priorto injection, and at 0, 15, 30 min, then 1 h, 2 h, 4 h, 8 h, 18 h or 30h, 42 h, 66 h, day 6 or 7 following the injection. Plasma or serum willbe collected and counted from each sample and the results will beexpressed as per cent of the injected radioactivity per L serum or bloodvolume. static spot view (SPOT) HEHR collimation 10 to 20 minacquisition time dual-window acquisition for scatter correction 128 ×128 × 16 matrix size SPECT HEHR collimation 6 degrees or 64 views instop and shoot, elliptical orbit mode 1 to 4 min/view (0.5 to 2 hacquisition time on a dual-headed camera) dual-window acquisition forscatter correction 64 × 64 × 16 matrix size whole-bodv sweep (SWEEP)high-energy, high-resolution (HEHR) collimation, 8 to 12 cm/min sweepspeed (20 to 25 min acquisition time) dual-window acquisition forscatter correction 256 × 1024 × 16 matrix size Imaging schedule: Imagingday SWEEP SPOT SPECT 0 X X 1, 2 X X X 5, 6 or 7 X X 9, 10, or 11 X X

2.1.2 Pharmacokinetics Modeling Blood time-activity curves from serialblood samples and from ROI's around sequential SPECT images of the heart(when available). This data will be fitted, together with the whole-bodyclearance kinetics, to a pharmacokinetic model of antibody distribution.Previously developed models have been used for this type of analysis,further details regarding the approach have been published.¹⁰⁶

2.1.3 Patient-specific dosimetry (3D-ID) The pharmacokinetic dataobtained from SPECT and planar imaging and blood sampling will becombined with anatomical imaging information (MR or CT) to estimate theabsorbed dose to tumor and selected normal organs that would be expectedfrom a therapeutic injection of ¹³¹I-8H9. The methodology for this hasbeen previously described.¹⁰⁷⁻¹¹⁵

2.2 Tumor volume determinations Tumor volumes will be determined from CTor MRI when available. Patients with known disease at other sites areimaged in additional areas. All CT images will be transferred fordisplay in 3D-ID; images collected at MSKCC will be transferreddigitally, film from other institutions will be scanned using a Lumisysdigital film scanner. Using 3D-ID, the consulting radiologist willreview the images with the research technician. The research technicianwill then draw contours around the tumor regions; the contours will bereviewed by the consulting radiologist and adjusted, as needed. In somecases, disease may be represented by a collection of very small positivenodes; in those cases a contour around the group will be drawn and usedin the volume assessment. Volume determination using 3D-ID is performedby summing the areas of regions that have been defined by the user onall slices making up the tumor. This general approach has beenpreviously validated for CT. Although potentially labor-intensive, sucha tumor outline-specific method is significantly more accurate thantechniques based upon greater and minor diameters (i.e., ellipsoidalmodels)). The errors associated with CT-based volume estimation and thefactors influencing these errors have been examined and will beconsidered in the volume determinations described above. A reliabletotal-body tumor burden will not be achievable for all patients, eitherbecause of the small volume of disease, or for cases in which lesionsdetected by SPECT are not visible by CT.

2.3 Red marrow dosimetry Bone marrow dosimetry will be performedaccording to the recommended guidelines, described in the AAPMrecommendations,¹¹⁶ i.e. blood time-activity curves will be multipliedby the appropriate factor (0.2-0.4) to derive marrow time-activitycurves and absorbed dose to red marrow. S-Factors provided in MIRDOSE 3will be used for the calculations. This data will be compared withdirect measurement of the marrow activity from ROI's drawn over marrowcavities on SPECT images. The quantitative capability of SPECT willallow us to verify the accuracy of bone marrow dosimetry determined fromactivity levels, and the rate of antibody clearance from marrow, fromthe standard analysis of serial blood samples.

2.4 Three-dimensional dosimetry To perform 3D dosimetry, it is firstnecessary to register a set of nuclear medicine images (SPECT),depicting the radiolabeled antibody distribution to an anatomicalimaging modality (CT or MRI). We have extensive experience with theclinical implementation of the Pelizzari and Chen method.¹¹⁷ Thistechnique requires that the user delineate the same surface on both setsof imaging modalities. When necessary, a SPECT transmission study isperformed to obtain the appropriate surface. The program attempts tomaximize the correlation of a set of several hundred points on thesurface as identified on one scan (the “hat”), with a solid model of thesame surface derived from the other scan (the “head”). A non-linearleast-squares search is used to minimize the sum of the squares ofdistances from each “hat” point to the nearest point on the “head”surface. The coordinates of the “hat” are translated, rotated and scaledto provide the best fit. Users may control which parameters are variedduring the search. The final set of transformations are then used toconvert the coordinates of one image into those of the other. Phantomstudies indicate that the Pelizzari and Chen technique for registrationof SPECT to CT is accurate to within 3mm. The Nuclear Medicine Serviceat MSKCC has performed such registration for over 100 patient studies.The Pellizari and Chen package has also been used for thoracic andabdominal study registration by Chen and his collaborators at theUniversity of Chicago (personal communication). Both the Chicago groupand us have also included contours for liver and/or spleen along withthe body contours. This further improves registration by providing morecontours for the minimization algorithm. In some cases, a radioactiveband has also been used as an aid to registration.¹¹⁷ We are currentlycomparing this method with alternative algorithms for image registrationfor the whole images.¹¹⁷⁻¹²⁰

Correlated serial SPECT images can be used to determine cumulativeactivity distributions by fitting and integrating an exponential uptakeand/or clearance to the specific activity within an ROI over the tumoror organ. The variation in activity within individual voxels can betaken into account, through a weighted sum of the counts/activity withinthe corresponding voxel over time. Given such a distribution of thecumulated activity, a software package, 3D-ID, has been developed, tocalculate the dose distribution. Target contours are drawn onside-by-side enlarged SPECT and CT/MR image slices that are selectedfrom a scrollable image display. Contours drawn in one modalitysimultaneously appear in the other. The user may switch betweenmodalities by positioning the cursor in the appropriate window. Thisprovides for the simultaneous use of both imaging modalities to definetumor (e.g. using SPECT) and normal organ (using CT/MR) contours. Thedose to all voxels within the target volume is obtained by convolvingthe activity distribution with a point kernel table of absorbed doseversus distance. Patient-specific S-factors may be calculated bydefining source organ contours and assigning unit activity to all voxelswithin each source. The “dose” to a given target is thus thepatient-specific S-factor. Dose histograms and patient-specific organand tumor S-factors generated using 3D-ID in combination with SPECT willprovide important information in understanding tumor response and organtoxicity in radioimmunotherapy.

Photon dose kernels for 14 radionuclides of interest in internal emittertherapy have been recently published.¹¹². Explicit expressions ofradionuclide photon dose kernels, necessary for three-dimensionaldosimetry, were not previously available. We recently described theoverall structure and methodologies of a software package forthree-dimensional internal dosimetry (3D-ID) calculations.^(107,113) Aseries of software modules that address the logistical issues ofperforming patient-specific three-dimensional dosimetry were detailed.Software tools have been developed to combine images from differentmodalities, define regions-of-interest using available multi-modalitydata and identify source and target volumes for dosimetry. Apoint-kernel based dosimetry calculation has been implemented andseveral different approaches for displaying the spatial distribution ofabsorbed dose in a biologically pertinent manner were also described.The dose calculation, itself, was carried out in a separate module, sothat different calculation schemes including Monte Carlo, may be usedwith 3D-ID.

2.5 Anticipated Results

The major sources of error in carrying out absorbed dose calculationsare: 1. Inaccuracies in imaging-derived activity concentrationestimates. 2. Mismatch between standard anatomy (used for dosimetrycalculations) and individual patient anatomy. 3. Assumption ofuniformity in the spatial distribution of radioactivity on both a micro(mm to mm) and macro (cm) scale. When applying conventional (MIRDCommittee) approaches to estimating absorbed dose it is understood thatthe estimate is derived from a model which includes a certain number ofassumptions. This approach has been sufficient in estimating doses fordiagnostic applications wherein typical doses are already far belowtoxicity. An objective of radioimmunotherapy, however, is to treat tonormal organ tolerance. In such a scenario, accurate, patient-specificdosimetry is critical. The dosimetry methodologies that will be used inthis proposal address point 2 and a portion of point 3; dosecalculations are performed for individual patient geometries and thespatial distribution of radioactivity in tumor or normal organs isaccounted for on a macroscopic (cm) scale. In the past using planarimaging kinetics to project the kinetics of the spatial distribution hadadditional pitfalls. Although SPECT-based activity determinations are astep forward, we expect these inaccuracies in imaging derived activityto be further reduced when I-124-8H9 Postron emission tomography isused. This is an area of active development at Memorial Sloan Ketteringin the last decade.¹²¹

Conventional dosimetry yields estimates of the absorbed dose, averagedover a normal organ or tumor volume. The methodology implemented in thisproposal will yield the spatial distribution of absorbed dose as isodosecontours, overlayed upon a 3-D CT image set. This makes it possible toevaluate the anatomical distribution of absorbed dose to tissues andfrom this, assess the potential impact in terms of toxicity. Forexample, the dose to surrounding tissue from activity that hasconcentrated in a tumor contained within a normal organ can be obtainedby this means.

2.6 Interpretations and Implications

The average absorbed dose to a tumor may not reflect potentialtherapeutic efficacy and tumor shrinkage. That portion of a tumor volumereceiving the lowest absorbed dose will lead to treatment failureregardless oft he dose delivered to other regions of the tumor volume.The 3D-ID software package provides detailed information regarding thespatial distribution of absorbed dose within a target volume. Thisinformation is depicted as dose-volume histograms, wherein the fractionof tumor volume receiving a particular absorbed dose is plotted againstabsorbed dose. Using such information it will be possible to betterassess the likelihood of tumor control. For example, if the average doseover a tumor volume is 2 to 3 Gy and a small region within this volumereceives only 0.1 Gy, then treatment will be unsuccessful.

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Ewing's sarcoma is a childhood tumor characterized by a t(11,22) in mostpatients. Because survival remains suboptimal with standard therapy,many patients receive autologous stem cell transplant and trialsinvestigating adoptive transfer of autologous T cells in the context ofimmune therapy are underway. However, approximately 50% of patients withadvanced disease have PCR detectable disease in peripheral blood and/orbone marrow and administration of contaminated autologous cellpreparations may contribute to disease relapse. To date, there is noreported method for purging contaminated hematopoietic cell populationsof Ewing's Sarcoma. 8H9 is a mouse monoclonal IgG1 antibody previouslyreported to react with 21/21 Ewing's sarcoma/PNET tumors (Proc ASCO17:44a, 1998). Peripheral blood T cell and B cell populations and CD34+cells from bone marrow analyzed by flow cytometry for binding of 8H9were negative. We sought to use magnetic bead immunoselection of 8H9labeled cells to purge peripheral blood cell populations contaminatedwith Ewing's sarcoma. Using real-time quantitative nested PCR withLightcycler, we monitored purging efficiency by evaluation of t(1,22) byRT-PCR. Contaminated specimens were labeled with 8H9 and incubated withrat anti-mouse IgGI magnetic beads. The sample was then run over aMiltenyi Variomax negative slection column. Recovery was approximately70%. RNA was extracted from 10e7 cells from pre and post purge cellpopulations. Real-time quantitative PCR was performed with a level ofsensitivity to one tumor cell in 10e5 normal cells. We demonstrated atleast a two-log reduction of tumor in preparations contaminated at aratio of 1:10 normal PBMC and 1:10e3 normal PBMC. Further studiesevaluating efficacy in clinical samples are underway. These resultsdemonstrate a potential new approach for purging contaminated patientsamples to be used in the context of autologous bone marrow transplantand/or immunotherapy trials for Ewing's sarcoma.

Immunomagnetic Purging of Ewing's Sarcoma from Blood and Bone Marrow:Quantitation by Real-Time PCR

The propensity for hematogenous spread of Ewing's sarcoma and theresulting contamination of autologous cell preparations complicates theuse of cellular therapies in this disease. To date, there has been noreported method for purging marrow and other cellular products ofEwing's sarcoma. In this paper, we introduce monoclonal antibody 8H9,which showed binding by flow cytometry to 9/9 Ewing's sarcoma cell linesstudied. Binding to lymphocytes and bone marrow progenitor cells wasnegative. In order to test whether 8H9 could be used for immunomagneticbased purging, normal PBMCs or bone marrow cells were artificiallycontaminated with varying amounts of Ewing's sarcoma. Quantitative PCRor t(11;22) was shown to accurately measure the level of contaminationwith a sensitivity of 1:10⁶. Samples were then purged using the MiltneyiVariomax negative selection system selecting for monoclonal antibody 8H9bound cells. A 2 to 3-log reduction in tumor burden was consistentlyobserved following immunomagnetic selection. In clinical non-mobilizedapheresis studied, Ewing's contamination ranged between 1:10⁵-1:10⁶.Therefore 8H9 based purging of clinical samples is predicted to resultin a contamination level which is below the limit of detection bysensitive quantitative PCR. These results demonstrate a potential newapproach for purging contaminated patient samples to be used in thecontext of autologous bone marrow transplant and/or immunotherapy trialsfor Ewing's sarcoma. (Mmerino@pol.net)

Current concepts hold that Ewing's sarcoma is a systemic disease fromthe time of onset as demonstrated by the observation that over 90% ofpatients with clinically localized disease will recur distantly iftreated with local measures alone[Jaffe, 1976 #49]. Indeed, thegenerally accepted factor responsible for the recent improvement insurvival observed in patients with clinically localized disease iscontrol of hematogenously disseminated micrometastasis via neoadjuvantmulti-agent chemotherapy¹. Recently, the use of sensitive molecularmonitoring to detect circulating Ewing's sarcoma cells has confirmedhematogenous dissemination in a substantial number of patients withEwing's sarcoma. West et al² found a 25% incidence of translocation(11;22) positivity in the peripheral blood or bone marrow in patientswith clinically localized disease, and higher rates have been observedin other series³ and in patients with overt metastatic disease.^(3, 4)Interestingly, in the reports by de Alava and Toretsky, evidence forpositivity in peripheral blood persisted following initiation ofchemotherapy suggesting that ongoing dissemination may occurintermittently throughout treatment protocols.

In an attempt to improve survival in high-risk patients with Ewing'ssarcoma, several groups have studied the use of high dose chemotherapyfollowed by bone marrow or peripheral stem cell transplantation.^(5-17.)Up to a 40% survival in poor risk patients has been reported after highdose therapy followed by autologous stem cells in contrast to historicalsurvival rates of 0-20% with chemotherapy/radiation therapyalone^(5, 6). One factor complicating the use of autologous stem cellproducts in therapy of Ewing's sarcoma is the propensity forhematogenous dissemination with resultant contamination of stem cellproducts. In one report, despite CD34 based positive selection forprogenitor cells, autologous peripheral blood progenitor preparationswere shown to contain EWS/FLI1 translocation positive cells in 54% ofsamples evaluated⁴. While the true clinical impact of contaminatingtumor cells in autologous products remains unclear, genetically markedtumor cells residing in autologous bone marrow have been shown to bepresent at disease relapse in patients with neuroblastoma andAML^(18, 19). Similar concern regarding the potential for autologouscell preparations to contribute to disease recurrence arise in thecontext of immune based therapy trials which are currently beingundertaken and involve the transfer of autologous T cells harvestedprior to the initiation of therapy²⁰.

To date there has been no method reported for purging autologoushematopoietic cells of Ewing's sarcoma. In this report, we introduce amonoclonal antibody based purging technique which allows us to reducethe tumor burden in contaminated bone marrow or peripheral bloodspecimens by two to three logs which is predicted to be below the limitof detection of PCR positivity in the vast majority of clinicallycontaminated specimens.

Materials and Methods

Monoclonal Antibody Production (Memorial Sloan-Kettering Cancer Center)

Cell Preparations

Peripheral Blood Mononuclear Cells: PBMCs used in tumor spikingexperiments were obtained by ficoll-based density gradient separation ofthe fresh buffy coat fraction of normal healthy donor blood unitsobtained at the Department of Transfusion Medicine, Clinical Center, NCIaccording to approved protocols. For analysis of T cell reactivity toanti-CD3 monoclonal antibody following purging, PBMCs were T cellenriched using a negative selection column (R & D Biosystems,Minneapolis) which results in a purity of approximately 80%. Patientapheresis samples analyzed for contamination were obtained as part ofNCI POB 97-0052 following informed consent. Leukapheresis procedureswere done using the CS3000 Plus (Fenwal Division, Baxter, Deerfield,Ill.) which processed 5-15 liters of blood volume. Countercurrentcentrifugal elutriation of the apheresis product was performed using aBeckman J-6M centrifuge equipped with a JE 5.0 rotor (BeckmanInstruments, Palo Alto, Calif.) in HBSS without magnesium, calcium andphenol red (BioWhittaker, Walkersville, Md.) at a centrifuge speed of3000 rpm (1725×g)2 ²¹. Cell fractions (450-550 ml each) were collectedat flow rates of 120, 140, and 190 ml/min. during centrifugation and at190 ml/min. with the rotor off (RO). The first two fractions aretypically enriched for lymphocytes while the last two fractions areenriched for monocytes. All fractions were cryopreserved in 10% DMSO(Cryoserv, Research Industries, Salt Lake City, Utah), RPMI withpenicillin, streptomycin and L-glutamine and 25% fetal calf serum.

Progenitor Cells: CD34+ cells used for purging experiments were selectedusing the Miltenyi Variomax® direct isolation system (Miltenyi, Auburn,Calif.) from cryopreserved peripheral stem cells from a Ewing's sarcomapatient obtained for therapeutic use at Children's National MedicalCenter, Washington, DC according to approved protocols and followinginformed consent. Stem cells were used for research purposes after thepatient's death. These cells were not positive by RT-PCR for Ewing'ssarcoma and were therefore artificially contaminated for the purgingexperiments. Non-CD34 selected bone marrow used for purging experimentsand enriched CD34+ populations used in the CFU assay were obtained fromfresh human marrow harvested from normal volunteers according toapproved protocols and following informed consent (PoieticsLaboratories, Gaithersburg, Md. The mononuclear fraction was obtained byficoll-based density gradient separation, and subsequently enriched forCD 34+ cells by the Miltenyi Variomax® (Miltenyi, Auburn, Calif.) directCD34 selection system.

Tumor Cell Lines: Ewing's sarcoma cell lines used for screening includedTC71, 5838, RD-ES, CHP100, A4573 which have been previously reported²²and JR and SB which are cell lines derived from patients treated at theNational Cancer Institute which have also been previously reported²². LGwas a cell line derived from a patient with isolated intrarenalrecurrence of Ewing's sarcoma treated with resection at the Universityof Maryland.

Flow Cytometry Analysis

Flow cytometric analysis was performed using the Becton-DckinsonFacsCalibur machine. Briefly, fluorescence data were collected using a3-decade log amplification on 10,000 viable gated cells as determined byforward and side light scatter intensity. Monoclonal antibodies used forimmunofluorescence were: MoAb 8H9, murine IgG1 isotype, goat anti-mouseIgG1-FITC, CD3-PE (S4.1), CD34-PE (581) Caltag (Burlingame, Calif.),CD99-FITC (TU12) (Pharmingen, San Diego, Calif.). For immunofluorescenceanalysis, cells were incubated with antibody at a concentration of 1μg/10⁶ cells for 20 minutes at 4°, followed by washing with PBS with0.2% human serum albumin and 0.1% Sodium Azide. For 8H9 and isotypestaining, this was followed by incubation with goat anti-mouse FITC for10 minutes at 4° C. followed by washing prior to analysis.

Immunomagnetic Purging

All cell products were spiked with tumor cells from the Ewing's sarcomacell line TC71 at the levels of contamination indicated for individualexperiments. For purging of CD34+ peripheral stem cells, a total of10×10⁶ were spiked. 1×10⁶ cells were analyzed for pre-purged andpost-purged PCR. For PBMC and non-CD34 selected bone marrow specimens,30-80×10⁶ cells were spiked with TC71 with 1×10⁶ cells analyzed forpre-purged and post-purged PCR. For purging, cells were incubated at 4°C. with monoclonal antibody 8 H9 at a concentration of 1 ug/10⁶ totalcells for 20 minutes and washed with buffer (PBS, 0.5% BSA, 2 mM EDTA).Cells were then incubated with rat anti-mouse IgG1 magnetic beads(Miltneyi, Auburn, Calif.) at a ratio of 1:1 for 20 minutes at 4° C.Purging was accomplished using the Miltenyi Variomax® system wherein thesample is run over the Miltenyi (Auburn, Calif.) AS depletion columnwith a flower resistor of 24 G. Cells from the depleted fraction werethen washed with 3 cc buffer. The positively selected fractions of cellswas removed by releasing the column from the magnet and washing with 3cc buffer, and analyzed by PCR where indicated. In cases whereclonogenicity of the positive fraction was evaluated, the positivefraction was pelleted and resuspended in RPMI with 10% FCS, L-glutamine(4 uM), penicillin (100 u/ml), and streptomycin (100 ug/ml), and placedin an incubator at 37° C. with 5% CO₂ for 5 days.

Conventionial PCR

For analysis of contamination of patient apheresis fractions, RNA wasextracted from 20-50×10⁶ cells using TRIzol Reagent (Life Technologies,Rockville, Md.) or guanidinium isothiocynate/CsCl method²³. After cDNAwas generated from 250 ng RNA using a random hexamer, PCR was performedwith Perkin Elmer GeneAmp PCR system 2400 using ESPB1 and ESBP2 primersand the following conditions: 40 cycles 95° C. 30 s, 60° C. 30 s, 72° C.30 s followed by 72° C. for minutes. To assess the integrity andquantity RNA, a PCR reaction with GAPDH primers was performed for eachpatient sample. 10 ul of each PCR product were run on 1.3% TBE agarosegel and transferred to a nylon membrane. A [³²P]γ-ATP 20-meroligonucleatide probe was generated using T4 polynucleotide kinase. Themembrane was hybridized using ExpressHyb Hybridization Solution(Clontech, Palo Alto, Calif.) according to the manufacturer'sinstructions. The membrane was then exposed to Kodak Xomat film (Kodak,Rochester, N.Y.) for 24-144 hours.

Real-Time Quantitative PCR

Real-time quantitative PCR was performed using the Lightcycler®Instrument (Roche Molecular Biochemicals, Indianapolis, Ind.). RNA wasextracted from 10×10⁶ cells from all samples except for the CD34+population in which 1×10⁶ cells were used. The Trizol® phenol/chloroformextraction or RNA-easy columns (Qiagen, Valencia, Calif.) were used. The1^(st) Strand Synthesis kit (Roche, Indianapolis, Ind.) was used togenerate cDNA from lug of RNA from each sample. PCR was then run on 5 ulof cDNA on the Lightcycler® instrument with primers ESBP1 and ESBP2 for40 cycles. In cases where nested PCR was performed, an initial 20 cyclesof PCR were carried out with the primer pair ESBP1-ESBP2 followed by 40additional cycles using 2 ul o f the product of the first reaction usingthe primer pair EWS 696-F11 1041 By conventional PCR, primer pairESBP1-ESBP2, and EWS 696-FLI 1041 generate fragments of 310 bp and 205bp respectively. Both sets of primers are outside the breakpoint of theEWS/FLI 1 translocation. In the initial evaluation of the quantitativePCR, both nested and non-nested Lightcycler® PCR products were confirmedby size using 1% TAE agarose gel with ethidium bromide (data not shown).Hybridization probes spanning the EWS/FLI 1 breakpoint were used todetect target template in the Lightcyler reaction. To provide a positivecontrol and to quantitate total amplified RNA, G6PD was amplified from 5ul of cDNA and analyzed using sequence specific hybridization probesG6PDHP1 and G6PDHP2. On all hybridization probes, the 5′ probe (HP1) waslabeled at the 3′end with Fluorescein, the 3′ probe (HP2) was labeled atthe 5′ end with Lightcycler Red 640 and phosphorylated at the 3 ′ end.Cycle crossing number was ascertained at the point in which all sampleshad entered the log linear phase. Cycle crossing number was used todetermine log cell concentration according to a standard curve. Thestandard curve was generated by amplifying 5 ul of cDNA derived from lugof RNA from 10×10⁶ normal PBMCs spiked with TC71 tumor cells atdecreasing concentrations from 1:10 to 1:10⁷. Sequences [³²P]γ Probe5′TACTCTCAGCAGAACACCTATG Primers ESBP1 5′ CGA CTA GTT ATG ATC AGA GCA 3′ESBP2 5′ CCG TTG CTC TGT ATT CTT ACT GA 3! EWS 696 5′ AGC AGC TAT GGACAG GAG 3′ FLI 1 041 5′ TTG AGG CCA GAA TTC ATG TT 3′ G6PD1 5′ CCG GATCGA CCA CTA CCT GGG CAA G 3′ G6PD 2 5′ GTT CCC CAC GTA CTG GCC CAG GACCA 3′ Lightcycler Hybridization Probes EWSHP1 5′ TAT AGC CAA CAG AGC AGCAGC TAC - F 3′ EWSHP2 5′ LC RED 640 - GGC AGC AGA ACC CTT CTT - P 3′G6PDHP1 5′ GTT CCA GAT GGG GCC GAA GAT CCT GTT G - F 3′ G6PDHP2 5′ LCRED 640 - CAA ATC TCA GCA CCA TGA GGT TCT GCA C - P 3′OKT3 Mediated Proliferation of Purged T Cell Specimens

1×10⁸ CD3 enriched cells were contaminated with Ewing's sarcoma at alevel of 1:10³. Cells from pre-purged and post-purged samples were addedin triplicate to a 96 well plate at a concentration of 2×10⁵ cells/wellcontaining decreasing concentrations of plate bound anti-CD3 antibodyOKT3 (Ortho Biotech Inc., Raritan, N.J.) from 100 ug/ml to 3 ug/ml.Cells were incubated with 200 ul of RPMI with 10% FCS, L-glutamine,penicillin, and streptomycin for a 48 hours and then pulsed with 1 uCiof [3H] thymidine per well. Cells were harvested after 18 hours ofpulsing and ³H incorporation was enumerated using the TopCount NXT(Packard, Meriden Conn.). Subtracting background activity with mediaalone generated net counts.

CFU Assay

CD34+ cells were enriched from pre- and post-purged samples from freshhuman bone marrow using the Miltenyi® direct CD34+ progenitor isolationkit. 35×10⁶ bone marrow mononuclear cells from each sample were run overa positive selection (MS) column yielding a CD34+ enriched populationwith estimated purities of >70%²⁴. 1000 cells were plated in triplicatein methylcellulose media supplemented with recombinant cytokines(MethoCultGF+H4435, Stem cell Technologies, Vancouver, BC). CFUs werecounted after 14 days of culture.

Results

Monoclonal Antibody 8H9 binds all Ewing's Sarcoma Cell Lines Tested butnot Normal Lymphocytes or Hematopoietic Progenitors.

In order to identify a potential reagent that could be used to targetcontaminating Ewing's sarcoma cells, monoclonal antibodies induced viaimmunization with neuroblastoma were screened for cross reactivity withEwing's sarcoma. Monoclonal antibody 8H9 was observed to bind to 9/9Ewing's sarcoma cell lines evaluated (FIG. 1). The level of reactivitywas variable with some lines showing diminished levels of reactivitycompared to C D99 whereas two lines (SB and RD-ES), showed increasedreactivity compared to CD99. Importantly, lymphoid and hematopoieticpopulations showed no reactivity with 8H9 as shown in FIG. 2 a (CD3gated PBMC), and FIG. 2 b (CD34 gated bone marrow cells), whereas CD99showed significant binding to T cell populations.

Quantification of Ewing's Sarcoma Contamination Using Real-Time PCR ofArtificially Contaminated Specimens Accurately Quantitates TumorContamination with Sensitivity to 1:10⁶.

To study whether Immunomagnetic purging of marrow and peripheral bloodpopulations contaminated with Ewing's sarcoma could be quantitativelymonitored, we sought to devise an approach wherein variable levels ofcontamination could be quantified using RT-PCR. We began by artificiallycontaminating PBMC populations with a log based titration of Ewing'scontamination (e.g. 1:10-1:10⁷). The degree of contamination wasevaluated using real-time PCR. Using a non-nested PCR, we observedlinear relationships across four log levels of contamination, (FIG. 3a). However, the limit of detection for a non-nested PCR was 1 tumorcell in 10⁴ background cells. In an effort to increase the sensitivity,we also evaluated nested PCR, using an initial 20 cycles ofamplification followed by 40 cycles amplification with internal primers.With this approach, quantitative accuracy was lost for only the highestlevel of contamination, which likely began to plateau with the initial20 cycles (3b). However, quantitative accuracy was observed for levelsof contamination between 1:100 to 1:10⁶ was observed (FIG. 3 c). Because10×10⁶ starting cells were used in these experiments, we can estimatethat using the nested approach, amplification was accomplished from 10contaminating cells. This confirmed the utility of quantitative PCR toprovide an accurate quantitative assessment of tumor contamination witha level of sensitivity of one tumor in 10⁶ background cells, thusallowing measurements of the efficacy of 8H9 based approaches forpurging of Ewing's sarcoma cells.

MoAb 8H9 Based Immunomagnetic Purging Yields a 2 to 3-log Reduction inArtificially Contaminated Peripheral Blood and Bone Marrow Populations.

In order to purge hematopoietic progenitor populations of Ewing'ssarcoma, variably contaminated 8H9 incubated bone marrow or peripheralblood stem cell populations were run over a Variomax® negative selectioncolumn as described in methods. Non-nested PCR evaluation of non-CD34selected bone marrow from a healthy donor spiked with Ewing's sarcomacells at a level of 1:100 is shown in FIG. 4 a. These resultsdemonstrate a 2-log reduction in tumor following 8H9 based purging. Toevaluate the efficiency of 8H9 based purging with progenitorcontamination at lower levels and to assess the ability to purge CD34+selected cells, CD34+ selected cells from G-CSF mobilized peripheralblood were spiked at a level of 1:10³ and purged as shown in FIG. 4 b.Using the quantitative PCR, we observed a 3-log reduction in the levelof contamination following one run over the column.

In the next experiments, evaluation of the ability to purge contaminatedPBMC populations was undertaken. Similar to the results observed withCD34+ enriched peripheral blood stem cells, at least a 3-log reductionin contamination following 8H9 based purging of PBMCs contaminated at1:100 was attained as shown in FIG. 4 c. Evaluation of purging of PBMCscontaminated at a lower level (1:10³) is shown in FIG. 4 d where a 3-logreduction is again observed. In each of these experiments analysis ofthe positive fraction demonstrated PCR positivity confirming selectionof contaminating Ewing's cells (data not shown). To account for anyvariation from the expected uniform amounts of starting RNA or cDNA,G6PD, amplification was performed from each sample in a quantitativefashion. We observed a variation in crossing time (reflective ofstarting template) of less than 2% in all of the samples indicating alow degree of variation in starting template between samples andconfirming viable RNA and cDNA in the negative samples (data not shown).These results suggested that monoclonal antibody 8H9 may be a suitablecandidate for immunomagnetic based purging of contaminated blood, bonemarrow, and CD 34+ enriched progenitor populations specimens with thelikelihood for purging to PCR negativity being high if the level ofcontamination present in clinical samples is less than 1:10⁴.

Contamination of Non-Mobilized Patient Apheresis Fractions with Ewing'sSarcoma is Between 1:10⁵-1:10⁶.

In order to evaluate the degree of contamination typically observed inclinical specimens, we studied non-mobilized peripheral blood apheresisspecimens derived from patients treated on immunotherapy trials forEwing's sarcoma at our institution. We observed a 66 % (8/12) incidenceof t(11,22) PCR positivity in non-mobilized apheresis specimens acquiredfor use in immunotherapy protocols as analyzed by conventional PCR(Table 1). As shown in Table 1, all elutriated apheresis fractions wereobserved to contain tumor with variability across individual patients.When elutriated apheresis specimens from several patients atpresentation of metastatic Ewing's sarcoma were analyzed usingquantitative PCR, this level of contamination was estimated to bebetween 1:10⁵ and 1:10⁶ with similar levels of contamination sometimesobserved in multiple apheresis fractions. (FIG. 5). Patient A (toppanel) showed positivity of all fractions at levels of approximately1:10⁶. Patient B (middle panel) showed a level of contamination ofapproximately 1:10⁶ in the 120 ml/min (lymphocyte) fraction with noevidence for positivity in the 190 ml/min or rotor off (monocyte)fractions. Patient C (bottom panel) showed a level of contaminationbetween 1:10⁵ and 1:10⁶ in multiple fractions. In no instance have weobserved levels of contamination greater than 1:10⁴. Therefore, becauseclinical specimens contaminated with Ewing's sarcoma appears to be inthe range of 1:10⁵-1:10⁶, it is anticipated that reduction incontamination to at least 1:10⁷ following 8H9 based purging will beachievable in the vast majority of patients. TABLE 1 Contamination ofnon-mobilized apheresis fractions with Ewing's sarcoma as analyzed byconventional PCR. Patient Lymphocyte Fractions Monocyte Fractions Number120 ml/min 140 ml/min 190 ml/min Rotor Off 1 N/A Positive NegativePositive 2 Positive Positive Positive Positive 3 Positive NegativePositive N/A 4 Negative Negative N/A Positive 3 Negative NegativeNegative Negative 6 N/A Negative Negative Positive 7 Negative NegativeNegative Negative 8 Negative Negative Negative Negative 8 NegativePositive Positive Negative 10 Positive Positive N/A Positive 11 NegativeNegative Negative Negative 12 Negative Negative Negative PositivePositive indicates band hybridized with the EWS/FLI radiolabeled probe.Negative indicated no band was noted. N/A indicated that no RNA wasobtained for that fraction.8H9 Based Purging Does Not Adversely Affect Stem Cell or T CellFunction.

To further evaluate the clinical feasibility of this technique forpurging of bone marrow or PBSC autografts, we sought to confirm retainedproliferative and differentiating capacity in 8H9 purged bone marrowpopulations. We studied CFU formation following purging as an assay ofCD34 function. We compared CFU formation before and after purging inCD34 selected bone marrow cells cultured in methycellulose media withrecombinant cytokines before and after purging (FIG. 6). We observednormal colony numbers and morphology in both samples with no significantdifference between samples indicating that CD34+ progenitors remainfunctional following 8H9 based purging.

T Cell Proliferation is Unchanged Before and After Purging.

Because T cells can contribute to post chemotherapy immunereconstitution²⁵, we are currently utilizing autologous T cell infusionsharvested prior to initiation of chemotherapy in order to study effectson immune reconstitution. In order to study T cell function following8H9 based purging, we evaluated T cell proliferation following anti-CD3cross linking as a measure of T cell function. We compared T cellproliferation unmanipulated T cells and 8H9 based purged T cells. Asshown in FIG. 7, there was no difference in T cell proliferationelicited by plate bound OKT3 antibody at concentrations ranging from 100ug/ml to 3 ug/nl as measured by [3w] thymidine uptake indicating that Tcell proliferative capacity is retained following 8H9 based purging(FIG. 7).

Discussion

The contribution of contaminated autologous preparations to diseaserelapse following autologous SCT in solid tumor patients is not fullyknown. Rill and Brenner et al. have shown than in certain solid tumors,tumors contaminating autologous grafts are tumorigenic and present atrelapse^(18, 19). In a disease such as Ewing's sarcoma, which has beenshown to have a high degree of hematogenous spread, this becomes animportant issue in the context of therapies which utilize autologouscells. In high-risk patients, survival after high dose chemotherapyfollowed by stem cell rescue continues to be suboptimal with the mostcommon cause of death due to disease relapse. Contamination ofautografts with subsequent survival and clonogenic growth of tumorpost-infusion cannot be excluded as contributing to this poor prognosis.In addition to the medical consequences of the administration ofcontaminated products to patients, psychologically there is reluctanceon the part of patients and their families to receive contaminatedproducts. It follows, therefore, that if a purging method was available,its evaluation for use in patients receiving autologous products iswarranted.

An ideal purging method should target only tumor cells and show nobinding to normal cell populations. The identification of such a tumorspecific antigen has historically posed a challenge in Ewing's sarcoma.While CD99 typically shows high expression on Ewing's sarcoma cells, itis also expressed on T cells (FIG. 2 a) and CD 34 stem cells²⁶, makingit unsuitable for purging hematologic products. Monoclonal antibody 8H9was initially developed due to its reactivity with neuroblastoma and wassubsequently reported to react with 19/19 fresh Ewing's sarcoma/PNETtumor confirming that 8H9 reactivity is not limited to established celllines²⁷. Our results (FIG. 1) confirmed this reactivity in all Ewing'scell lines evaluated. Since this antibody showed no reactivity with Tcells and CD34+ cells, it was ideally suited for purging. Indeed, wedemonstrated a 2-3 log reduction in all experiments following one runover the negative selection column. In the clinical setting ofautologous stem cell transplant, the combination of positive selectionfor CD34+ cells, which results in an approximate 2-log passive depletionof tumor^(28, 29), followed by 8H9 purging of tumor cells would beexpected to result in up to 5 logs of depletion, which is predicted tobe well below the limit of detection using currently availabletechniques. Further, even in the setting of autologous T celltransplantation, as potentially used in the context of immunereconstitutive therapies²⁰, the use of 8H9 based purging with its 2-3log reduction will substantially diminish the tumor burden contained inautologous cellular products.

This is the first published report of 8H9 as a Ewing's reactivemonoclonal antibody. Interestingly, 8H9 also shows reactivity withseveral rhabdomyosarcoma and osteosarcoma cell lines (data not shown).This introduces the exciting possibility of a sarcoma specific antibodywith potential applications in immune directed therapy. In addition,identification and characterization of the tumor specific epitope whichbinds to 8H9 could offer important insight into the biology of thesetumors. These studies are currently underway. Further, during the courseof the studies reported here, we sought to evaluate in a general sense,the function of sarcoma cells selected with 8H9. We observed thatEwing's sarcoma cells positively selected using 8H9 retain theirclonogenic properties and are able to be maintained in cell culture.This property has the potential aid in the generation and study of tumorcell lines derived from patients with pediatric sarcomas, which iscurrently difficult in these tumors due to limitations of tumor size andsurgical accessibility of primary tumors. We are currently investigatingwhether Ewing's sarcoma cells derived from apheresis or bone marrowsamples in patients with metastatic disease which are positivelyselected and grown in culture could provide a ready source of tumorsamples for further biologic study.

RT-PCR is a powerfully sensitive tool for use in monitoring minimalresidual disease MRD³⁰. It remains unclear, however, whether evidence ofsmall amounts of residual tumor by molecular analysis is predictive forrelapse in solid tumors and data in the literature is conflicting. deAlava et al. evaluated MRD in Ewing's sarcoma patients and showed acorrelation between PCR positivity and disease relapse. In this reporthowever, some patients remained PCR positive without disease relapse³.Using real-time PCR, it is now possible to quantitate starting templateand compare starting template amount between samples obtained atdifferent timepoints. Real-time quantitative PCR has been used as a toolto monitor MRD in leukemia patients^(31, 32) and may be useful inevaluation of disease response³³ and in predicting relapse in patientsby the detection of increasing levels of tumor specific transcript.

This is the first report of the use of real-time quantitative PCR usedto detect and quantify Ewing's sarcoma transcript. It is possible thatquantitative PCR could allow for further identification of patients witha high risk of relapse by detection of increasing amounts of Ewing'stranscripts over time. However, because contamination of peripheralblood by solid tumors is likely to be relatively low (in the range of1:10⁵-1:10⁶ in this series), the sensitivity of this analysis must bevery high in order to allow for the detection of very low levels ofcirculating tumor in patients with solid tumors. The level ofsensitivity of our technique reached 1 Ewing's sarcoma cell in 10⁶normal cells with nested PCR from 10×10⁶ cells. It is possible that thelevel of sensitivity would be even higher if higher cell numbers wereevaluated since this method appears capable in our hands of amplifyingproduct from 10 contaminating cells. Tumor enrichment using positiveselection is another method to increase sensitivity of tumor detection.The positive immunomagnetic selection procedure described in this paperfor purging could also provide a suitable approach for tumor enrichmentin for monitoring MRD or even in contributing to making the correctdiagnosis at the time of initial presentation with metastatic disease.Indeed, cells eluted from the column were positive by PCR analysis,demonstrating the feasibility of this technique for tumor enrichmentwhich would be predicted to increase the sensitivity of PCR detection ofcontaminating Ewing's sarcoma in patient samples. One caveat whichshould be noted is that the quantitative technique, relies on theassumption that the level of expression of t(11;22) is consistent amongcell lines and patient samples. This, may not be the case, however, andmay lead to under or over estimation of the absolute level of tumorburden when comparing patient samples to a standard curve. Suchlimitations would not preclude evaluation of changes in the level of PCRpositivity of an individual patient over time, wherein substantialchanges in the level of expression of t(11;22) may be less likely.

In this report we have demonstrated a purging technique that reducestumor burden in artificially contaminated products by at least 2-3 logs.This approach is predicted to substantially reduce the tumor burdencontained in autologous cellular products which are administered in thecontext of innovative therapies for Ewing's sarcoma. The demonstrationthat CFU assays on progenitor cells as well as CD3 induced T cellproliferation are normal after purging demonstrates no detrimentaleffects on normal progenitor cell and T cell function, making this apotentially feasible addition to autologous protocols. We conclude thatimmunomagnetic purging via negative selection using MoAb 8H9 warrantsevaluation in clinical trials for Ewing's sarcoma involving the use ofautologous products.

REFERENCES

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Desmoplastic small round cell tumor (DSRCT) is an aggressive, oftenmisdiagnosed neoplasm of children and young adults. It ischemotherapy-sensitive, yet patients often relapse off therapy becauseof residual microscopic disease at distant sites: peritoneum, liver,lymph node and lung. Strategies directed at minimal residual disease(MRD) may be necessary for cure. Monoclonal antibodies selective forcell surface tumor-associated antigens may have utility for diagnosisand therapy of MRD, as recently demonstrated in advanced-stageneuroblastoma (JCO 16: 3053, 1998). Using immunohistochemistry, westudied the expression of two antigens: (1) G_(D2) using antibody 3F8and (2) a novel antigen using antibody 8H9, in a panel of 36 freshlyfrozen DSRCT. G_(D2) is a disialoganglioside which is widely expressedamong neuroectodermal tumors as well as adult sarcomas. 8H9 recognizes asurface 58 kD antigen expressed among neuroectodermal, mesenchymal andepithelial tumors with restricted expression on normal tissues. 27 of 37tumors (73%) were reactive with 3F8, and 35 of 37 (95%) with 8H9. BothG_(D2) and the 58 kD antigen were found on tumor cell membrane and instroma. In general, immunoreactivity was stronger and more homogeneouswith 8H9 than with 3F8. These antigens are potential targets forimmunodiagnosis and antibody-based therapy of DSRCT.

Desmoplastic small round cell tumor (DSRCT) is an aggressive,ill-understood tumor affecting children and young adults. It ischaracterized clinically by widespread abdominal serosal involvement,metastasizes to peritoneum, liver, lungs and lymph nodes, and isassociated with a poor prognosis (Gerald et al., 1991). Histologically,it consists of small, undifferentiated round cells surrounded by anabundant desmoplastic stroma. Immunohistochemically, the coexpression ofepithelial, neural and muscle markers is typical (Ordonez et al., 1993).DSRCT is associated with a specific chromosomal translocation,t(11;22)p13;q12). The fused gene product aligns the NH2 terminal domainof the EWS gene to the zinc finger DNA-binding domain of the WT1 geneand is diagnostic of DSRCT (Ladanyi et al., 1994). This fusion resultsin the induction of endogenous platelet derived growth factor-A whichstimulates fibroblast growth and may contribute to the unique fibrosisobserved with this tumor (Lee et al, 1997). Further evidence ofupregulation of growth factors includes the reported expression ofIGF-II, PDGF-α receptor and IL-11 in DSRCT (Froberg et al., 1999).

Although dramatic response to aggressive multimodality therapy has beendemonstrated in the patients with DSRCT (Kushner et al., 1996), manypatients relapse with recurrent local disease or distant metastases.Strategies aimed at eradication of MRD are, therefore, warranted in themanagement of patients with DSRCT. Monoclonal antibodies selective forcell surface tumor-associated antigens are potential candidates asrecently demonstrated in neuroblastoma where immune targeting of thediasialoganglioside GD2 has significantly improved long-term survival inpatients with stage 4 disease (Cheung et al., 1998). Few suchtumor-associated targets have been defined for DSRCT. We describe heretwo possible targets for such immunotherapy: GD2 targeted by themonoclonal antibody 3F8 and a novel tumor antigen recognized by themonoclonal antibody 8H9.

Materials and Methods

Tumor and Normal Tissue Samples

Frozen tumors from 37 patients with DSRCT were analyzed. Diagnosis wasconfirmed by hematoxylin and eosin assessment of paraffin-fixedspecimens.

Monoclonal Antibodies

The murine IgG₃ monoclonal antibody 3F8 was purified from ascites aspreviously described (Cheung et al., 1985). Using a similar technique,female BALB/c mice were hyperimmunized with human neuroblastoma.Lymphocytes derived from these mice were fused with SP2/0 mouse myelomacells line. Clones were selected for specific binding on ELISA. The 8H9hybridoma secreting an IgG₁ monoclonal antibody was selected. 8H9 wasproduced in vitro and purified by protein G (Pharmacia, Piscataway,N.J.) affinity chromatography.

Immunohistochemical Studies

Eight μm cryostat frozen tumor sections were fixed in acetone and washedin PBS. Immunohistochemical studies were performed as previouslydescribed (Kramer et al. 1996) Endogenous peroxidases were blocked in0.3% H₂O₂ in PBS. Sections were incubated in 10% horse serum (Gibco BRLGaithersburg, Md.) after blocking with avidin and biotin. Incubationwith purified 8H9 diluted in PBS to 2 μg/ml was carried out at roomtemperature for 1 hour. An IgG1 myeloma was used as a control (SigmaChemical, St Louis Mo.). Sections were incubated with a secondary horseanti-mouse biotinylated antibody (Vector Laboratories, Burlingame,Calif.) followed by incubation with ABC complex (Vector Laboratories,Burlingame, Calif.) and stained with Vector VIP peroxidase substrate(Vector Laboratories, Burlingame, Calif.) or DAB peroxidase substratekit (Vector Laboratories, Burlingame, Calif.). A 10% hematoxylincounterstain for 2 minutes was used.

Staining was graded as positive or negative and homogenous orheterogenous reactivity noted.

Results

Clinical Profile

Of the 37 patients studied, 32 were male and five female. Age atdiagnosis ranged from 13 to 46 years (median 18 years). All receivedtreatment with an aggressive multimodality regimen includingdose-intensive chemotherapy.

Immunoreactivity

Tumor sections from 3 7 patients were tested for the expression ofG_(D2) and the antigen recognized by 8H9 by immunohistochemistry. 27 of37 (73%) tested positive for G_(D2). (Table 1). Most tumors had strongimmunoreactivity (>1+). Immunoreactivity was seen homogeneously in mosttumors and was localized to the cell membrane (FIG. 1). Intense stromalstaining was marked in all tumors studied. TABLE 1 Immunoreactivity of3F8 and 8H9 with DSRCT No. Reactivity No. Homo- Hetero- Marker tested 01+ 2+ 3+ pos. (%) geneous geneous G_(D2) 36 10 10 12 4 26 (72) 19 7Antigen 36 2 9 17 8 34 (94) 32 2 8H9

35 of 37 (95%/o) tumors tested positive for 8H9. Immunoreactivity had acharacteristic cell membrane localization and was homogeneous in almostall tumors (FIG. 2). Immunoreactivity was more strongly marked than thatwith 3F8. Equally strong stromal staining was seen.

Clinicopathologic Correlation

In this group of highly aggressive disseminated tumors, there was nocorrelation between outcome and the expression of either G_(D2) or the8H9 antigen (Table 2) TABLE 2 G_(D2) and Antigen 8H9: Correlation withoutcome G_(D2) positive 8H9 positive Expired* 10/17 16/17 Survivors <18mo since diagnosis 11/14 13/14 Survivors >18 mo since diagnosis 5/5 5/5*1 patient died of treatment-related toxicityDiscussion

The clinicopathological spectrum of DSRCT continues to be furtherdefined since the initial series was reported in 1991 (Gerald et al.,1991). Chemosensitivity to doxomibicin and alkylator-based chemotherapyhas been reported (Gonzalez-Crussi et al., 1990). Prolonged survival inresponse to an aggressive multimodality regimen including high-dosechemotherapy, radiation and surgery has also been reported (Kushner etal., 1996). However, most patients succumb to recurrent local disease ormetastases to peritoneum, liver, lymph nodes, or lung. Relapses can belargely attributed to the failure of eradication of MRD. Alternativetherapeutic strategies to target MRD are therefore warranted. One suchstrategy could be directed at the upregulated growth factorsparticularly PDGFA and related factors expressed on DSRCT (Froberg etal., 1999). Targeted immunotherapy utilizing monoclonal antibodies,which does not add to the toxicity of chemotherapy, is another approach.

DSRCT is characterized by the coexpression of epithelial, mesenchymaland neuroectodermal markers. Recent publications have defined theimmunohistochemical and molecular make-up of DSRCT (Ordonez, 1998;Gerald, 1999). However, most of the markers identified cannot be used astargets for antibody mediated immunotherapy either due tocrossreactivity with normal tissues or inaccessibility to monoclonalantibodies due to localization in the nucleus or cytoplasm. (Table 3).The most commonly expressed markers on DSRCT including desmin,cytokeratin, vimentin, epithelial membrane antigen and neuron-specificenolase are also widely expressed on normal tissues. The MIC2 antigenhas been reported to be expressed on 20-35% of DSRCT. However, unlikeEwing's sarcoma family of tumors, which have membrane localization,immunoreactivity in DSRCT is primarily cytoplasmic (Gerald et al, 1998).MOC31, a monoclonal antibody that recognizes epithelial glycoprotein 2(EGP-2) has been shown to be reactive with most DSRCT tested (Ordonez,1998). EGP-2 is overexpressed on epithelial tumors, but is also presenton normal epithelial cells (de Leij et al, 1994). Antibodies directedagainst the WT1 protein have strong, specific, nuclear immunoreactivitywith almost all DSRCT tested (Gerald et al, 1998) TABLE 3 Previouslyreported antigens on DSRCT Antigen Localization CrossreactivityIntermediate filaments Desmin cytoplasm skeletal, cardiac & smoothmuscle Vimentin cytoplasm mesenchymal tissues Keratin cytoplasmepithelial cells Epithelial antigens Epithelial membrane antigencytoplasm epithelial cells Epithelial glycoprotein-2 cytoplasmepithelial cells Ber-Ep4 antigen cytoplasm epithelial cells Neuralantigens CD57 cytoplasm neural tissues Neuron-specific enolase cytoplasmneural tissues MIC-2 cytoplasm & lymph nodes, cell membrane epithelialcells WT1 protein Nucleus None PDGFA Cell membrane Endothelial cells,PDGF-αreceptor Cell membrane hematopoeitic cells Endothelial cells,hematopoeitic cells

The reported expression of neuroectodermal antigens on DSRCT led us tostudy these tumors for the expression of G_(D2): a disialogangliosidewhich is expressed on other small blue round cell tumors such asneuroblastoma, small cell lung cancer, melanoma and osteosarcoma (Heineret al., 1987) as well as on adult soft tissue sarcomas (Chang et al.,1992). G_(D2) is a safe target for immunotherapy based on clinicaltrials of the anti-G_(D2) antibody 3F8 in patients with neuroblastomatissues of the nervous system (Cheung et al., 1998). Serum G_(D2) doesnot interfere with the biodistribution of specific antibodies and theantigen is not modulated from the cell surface upon binding byantibodies. Successful targeting of the monoclonal antibody 3F8 toG_(D2) was previously demonstrated in neuroblastoma (Yeh et al., 1991)and small cell lung cancer (Grant et al., 1996). 3F8 has also shownefficacy in clinical trials in patients with neuroblastoma (Cheung etal., 1998b) and melanoma (Cheung et al., 1987). Furthermore, 3F8appeared to induce long-term remissions in patients with Stage 4neuroblastoma. Reported side effects are short-lived and manageable(Cheung et al., 1998). In our study 72% of DSRCT tested wereimmunoreactive with the anti-GD2 antibody 3F8. Most tumors showedstrong, homogeneous reactivity localized to the cell membrane. (Table 1)(FIG. 1) DSRCT may be a putative tumor for in vivo antibody targetingwith 3F8. Alternatively, an anti-idiotypic vaccine approach can beutilized as has been suggested for neuroblastoma. 20 (Cheung et al,1994)

The monoclonal antibody 8H9 is a murine IgG₁ derived from mice immunizedwith neuroblastoma. It has been shown to have a broad expression onneuroectodermal, mesenchymal and epithelial tumors with limitedexpression on normal tissues. (data not shown). Its immunoreactiveprofile led us to u se i t for testing DSRCT. 95% of tumors testedpositive with DSRCT. Immunoreactivity with DSRCT was localized to thestroma and cell membrane FIG. 2) and for most tumors was intense andhomogeneous, and in general, stronger than that observed for GD2 (Table2).

The target antigen for 8H9 appears to be a novel 58 kD glycoprotein witha unique distribution on cell membranes of tumors of varying lineage,but restricted expression in normal tissues. This tissue distributionmakes it likely to be a unique antigen not previously described onDSRCT. The cell membrane localization of 8H9 allows it to be targeted bymonoclonal antibodies. 8H9 conjugated with I¹³¹ has been shown toradioimmunolocalize neuroblastoma and rhabdomyosarcoma xenografts inmice without significant crossreactivity with other organs. (data notshown).

In the therapy of DSRCT, strategies to eliminate minimal residualdisease are necessary to produce cures. Monoclonal antibody basedtherapy may augment aggressive multimodality therapy by targetingminimal residual disease without adding to toxicity. Our study hasidentified G_(D2) and antigen 8 H9 as two hitherto undescribed markersfor DSRCT, which can potentially be targets for differential diagnosisand immunotherapy.

REFERENCES

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J.Nucl.Med. 32: 769-76 (1991)    Fifth Series of Experiments    Anti-Idiotypic Antibody as the Surrogate Antigen for Cloning ScFv    and Its Fusion Proteins

ScFv provides a versatile homing unit for novel antibody-fusionconstructs. However, a reliable screening and binding assay is often thelimiting step for antigens that are difficult to clone or purify. Wedemonstrate that anti-idiotypic antibodies can be used as surrogateantigens for cloning scFv and their fusion proteins. 8H9 is a murineIgG1 monoclonal antibody specific for a novel antigen expressed on thecell surface of a wide spectrum of human solid tumors but not in normaltissues (Cancer Res 61:4048,2001) Rat anti-8H9-idiotypic hybridomas(clones 2E9, 1E12 and 1F11) were produced by somatic cell fusion betweenrat lymphocytes and mouse SP2/0 myeloma. In direct binding assays(ELISA) they were specific for the 8H9 idiotope. Using 2E9 as thesurrogate antigen, 8H9-scFv was cloned from hybridoma cDNA by phagedisplay. 8H9scFv was then fused to human-1-CH2-CH3 cDNA for transductioninto CHO and NSO cells. High expressors of mouse scFv-human Fc chimericantibody were selected. The secreted homodimer reacted specifically withantigen-positive tumor cells by ELISA and by flow cytometry, inhibitableby the anti-idiotypic antibody. The reduced size resulted in a shorterhalf-life in vivo, while achieving comparable tumor to nontumor ratio asthe native antibody 8H9. However, it could not mediateantibody-dependent cell-mediated or complement-mediated cytotoxicitiesin vitro.

1. Introduction

The ability to condense the binding site by genetic fusion of variableregion immunoglobulin genes to form scFv has greatly expanded thepotential and development of antibody-based targeted therapies (Bird etal., 1988; Huston et al., 1988; Winter and Milstein, 1991; George etal., 1994). Using phage display libraries, scFv can now be cloned fromcDNA libraries derived from rodents, immunized volunteers, or patients(Burton and Barbas III, 1994; Winter et al., 1994; Cai and Garen, 1995;Raag and Whitlow, 1995). The availability of hIg-transgenic andtranschromosomal mice will allow immunization schema or pathogens notfeasible or safe in humans. Construction of the scFv is the criticalfirst step in the synthesis of various fusion proteins, includingscFv-cytokine (Shu et al., 1993), scFv-streptavidin (Kipriyanov et al.,1995), scFv-enzyme (Michael et al., 1996), scFv-toxins (Wikstrand etal., 1995), bispecific scFv (diabodies) (Alt et al., 1999), bispecificchelating scFv (DeNardo et al., 1999), scFv-Ig (Shu et al., 1993),tetravalent scFv (Alt et al., 1999; Santos et al., 1999) andscFv-retargeted T-cells (Eshhar et al., 1993). ScFv-Ig constructs mimicnatural IgG molecules in their homodimerization through the Fc region,as well as their ability to activate complement (CMC) and mediateantibody dependent cell-mediated cytotoxicites (ADCC).

The construction of scFv requires a reliable antigen preparation bothfor panning phages and for binding assays. They often become arate-limiting step (Lu and Sloan, 1 999), particularly for antigens thatare difficult to clone or purify. Cell-based phage display (Watters etal., 1997), and enzyme linked immunosorbent assays (ELISA) whenoptimized, have been successfully applied as alternatives. However,subtle differences in the panning step can determine the success orfailure of phage display (Tur et al., 2001). For example, a reduction inwash pH is needed for scFv directed at ganglioside G_(D2) in order toreduce nonspecific adherence of phage particles (Tur et al., 2001).Moreover, phage binding assay may require membrane preparations towithstand the vigorous washing procedure.

Anti-idiotypic antibodies are frequently used as antigen mimics ofinfectious agents and tumor antigens (Thanavala et al., 1986; Wagner etal., 1997). When made as MoAb, they are ideal surrogates when the targetantigen is not readily available. The physico-chemical behavior ofimmunoglobulins as antigens in panning and binding assays is generallyknown and can be easily standardized. We recently described a noveltumor antigen reactive with a murine MoAb 8H9 (Modak et al., 2001).Given its lability and glycosylation, this antigen is difficult topurify. Here we describe the use of an anti-idiotypic antibody as asurrogate antigen for cloning a scFv derived from the 8H9 hybridoma cDNAlibrary, and for the selection of chimeric mouse scFv-human Fc fusionconstructs.

2. Materials and Methods

2.1 Animals

BALB/c mice were purchased from Jackson Laboratories, Bar Harbor, Me.Lou/CN rats were obtained from the National Cancer Institute-FrederickCancer Center (Bethesda, Md.) and maintained in ventilated cages.Experiments were carried out under a protocol approved by theInstitutional Animal Care and Use Committee, and guidelines for theproper and humane use of animals in research were followed.

2.2 Cell Lines

Human neuroblastoma cell lines LAN-1 was provided by Dr. Robert Seeger(Children's Hospital of Los Angeles, Los Angeles, Calif.), and NMB7 byDr. Shuen-Kuei Liao (McMaster University, Ontario, Canada). Cell lineswere cultured in 10% defined calf serum (Hyclone, Logan, Utah) in RPMIwith 2 mM L-glutamine, 100 U/ml of penicillin (Sigma-Aldrich, St. Louis,Mo.), 100 ug/ml of streptomycin (Sigma-Aldrich), 5% CO₂ in a 37° C.humidified incubator. Normal human mononuclear cells were prepared fromheparinized bone marrow samples by centrifugation across aFicoll-Hypaque density separation gradient. Human AB serum (GeminiBioproducts, Woodland, Calif.) was used as the source of humancomplement.

2.3 Monoclonal Antibodies

Cells were cultured in RPMI 1640 with 10% newborn calf serum (Hyclone,Logan, Utah) supplemented with 2 mM glutamine, 100 U/ml of penicillinand 100 ug/ml of streptomycin (Sigma-Aldrich). 3F8, an IgG3 MoAb raisedin a Balb/c mouse against human neuroblastoma, specifically recognizesthe ganglioside GD2. The BALB/c myeloma proteins MOPC-104E, TEPC-183,MOPC-351, TEPC-15, MOPC-21, UPC-10, MOPC-141, FLOPC-21, and Y5606 werepurchased from Sigma-Aldrich. MoAb R24 (anti-GD3), V1-R24, and K9(anti-GD3) were gifts from Dr. A. Houghton, OKB7 and M195 (anti-CD33)from Dr. D. Scheinberg, and 10-11 (anti-GM2) from Dr. P. Livingston ofMemorial Sloan Kettering Cancer Center, New York; and 5 28 (EGF-R) fromDr. J. Mendelsohn of MD Anderson, Houston, Tex. 2E6 (rat anti-mouseIgG3) was obtained from hybridomas purchased from American Type CultureCollection [ATCC] (Rockville, Md.). NR-Co-04 was provided by GeneticsInstitute (Cambridge, Mass.). In our laboratory, 5F9, 8H9, 3A5, 3E7,1D7, 1A7 were produced against human neuroblastoma; 2C9, 2E10 and 3E6against human breast carcinoma, and 4B6 against glioblastoma multiforme.They were all purified by protein A or protein G (Pharmacia, Piscataway,N.J.) affinity chromatography.

2.4 Anti-8H9 Anti-Idiotypic Antibodies

LOU/CN rats were immunized intraperitoneally (ip) with 8H9 (400 ug perrat) complexed with rabbit anti-rat serum (in 0.15 ml), and emulsifiedwith an equal volume (0.15 ml) of Complete Freund's Adjuvant (CFA)(Gibco-BRL, Gaithersburg, Md.). The 8H9-rabbit-IgG complex was preparedby mixing 2 ml (8 mg) of purified 8H9 with 4 ml of a high titer rabbitanti-rat precipitating serum (Jackson Immunoresearch Laboratories, WestGrove, Pa.). After incubation at 4° C. for 3 hours, the precipitate wasisolated by centrifugation at 2500 rpm for 10 minutes, and resuspendedin PBS. Three months after primary immunization, the rats were boostedip with the same antigen in CFA. One month later, a 400 ug boost of8H9-rabbit-anti-mouse complex was injected intravenously. Three daysafterwards, the rat spleen was removed aseptically, and purifiedlymphocytes were hybridized with SP2/0-Ag14 (ATCC). Clones selection wasbased on specific binding to 8H9 and not to control antibody 5F9, amurine IgG1. Repeated subcloning using limiting dilution was done.Isotypes of the rat monoclonal antibodies were determined by MonoclonalTyping Kit (Sigma-Aldrich). Rat anti-idiotypic antibody clones (2E9,1E12, 1F11) were chosen and produced by high density miniPERM bioreactor(Unisyn technologies, Hopkinton, Mass.), and purified by protein Gaffinity chromatography (Hitrap G, Pharmacia). The IgG fraction waseluted with pH 2.7 glycine-HCl buffer and neutralized with 1 M Trisbuffer pH 9. After dialysis in PBS at 4° C. for 18 hours, the purifiedantibody was filtered through a 0.2 um millipore filter (Millipore,Bedford, Mass.), and stored frozen at −70° C. Purity was determined bySDS-PAGE electrophoresis using 7.5% acrylamide gel.

The “standard” ELISA to detect rat anti-idiotypic antibodies (Ab2) wasas follows: Purified 8H9, or irrelevant IgG1 myeloma, were diluted to 5ug/ml in PBS and 50 ul per well was added to 96-well flat-bottomedpolyvinylchloride (PVC) microtiter plates and incubated for 1 hour at37° C. Rows with no antigen were used for background subtraction. Fillerprotein was 0.5% BSA in PBS and was added at 100 ul per well, andincubated for 30 minutes at 4° C. After washing, 50 ul duplicates ofhybridoma supernatant was added to the antigen-coated wells andincubated for 3 hours at 37° C. The plates were washed and aperoxidase-conjugated mouse anti-rat IgG+IgM (Jackson ImmunoresearchLaboratory) at 100 ul per well was allowed to react for 1 hour at 4° C.The plate was developed using the substrate o-phenylenediamine(Sigma-Aldrich) (0.5 mg/ml) and hydrogen peroxide (0.03%) in 0.1 Mcitrate phosphate buffer at pH 5. After 30 minutes in the dark, thereaction was quenched with 30 ul of 5 N sulfuric acid and read using anELISA plate reader.

2.5 Specificity by Direct Binding Assay

Fifty ul per well of purified mouse monoclonal antibodies or myelomaswere coated onto 96-well PVC microtiter plates at 5 ug/ml for 60 minutesat 37° C., aspirated and then blocked with 100 ul of 0.5% BSA fillerprotein per well. After washing and air-drying, the wells were allowedto react with anti-idiotypic antibodies. The rest of the procedure wasidentical to that described in the “standard” assay.

2.6 Specificity by Inhibition Assay

To further examine the specificity of these anti-idiotypic antibodies,inhibition of 8H9 immunofluorescent staining of tumor cells byanti-idiotypic antibodies was tested. Purified 8H9 and anti-G_(D2) MoAb3F8, (all 10 ug/ml in 0.5% BSA) were preincubated with variousconcentrations of anti-idiotypic antibodies for 30 minutes on ice beforereacting with 10⁶ cells of either GD2-positive/8H9 positive LAN-1(neuroblastoma) or GD2-negative/8H9-positive HTB-82 (rhabdomyosarcoma).The cells were then washed twice in PBS with 0.1% sodium azide andreacted with FITC-conjugated rat anti-mouse IgG (Biosource, Burlingame,Calif.) on ice for 30 minutes in the dark. The cells were washed in PBSwith azide, fixed in 1% paraformaldehyde and analyzed by FACScan(Becton-Dickinson, Calif.). The mean fluorescence was calculated and theinhibition curve computed.

2.7 Construction of scFv Gene

mRNA was isolated from 8H9 hybridoma cells using a commerciallyavailable kit (Quick Prep Micro mRNA Purification, Pharmacia Biotech)following the procedures outlined by the manufacturer. 5×10⁶ hybridomacells cultured in RPMI-1640 medium supplemented 10% calf serum,L-glutamine (2 mmol/L), penicillin (100 u/L) and streptomycin sulphate(100 ug/ml) were pelleted by centrifugation at 800×g and washed once inRNase-free phosphate buffered saline (pH 7.4). The recentrifuged cellswere lysed directly in the extraction buffer. Poly(A)-RNA was purifiedby a single fractionation over oligo (dT)-cellulose and eluted fromoligo (dT) cellulose in the elution buffer. The mRNA sample wasprecipitated for 1 hour with 100 ug glycogen, 40 ul of 2M potassiumacetate solution and 1 ml of absolute ethanol at −20° C. The nucleicacid was recovered by centrifugation at 10,000×g for 30 min. The samplewas evaporated until dry, and dissolved in 20 ul RNase-free water.

ScFv gene was constructed by recombinant phage display. 5 ul of mRNA wasreversely transcribed in a total volume of 11 ul reaction mixture and 1ul dithiothreitol (DTI) solution for 1 hour at 37° C. For the PCRamplification of 8H9 immunoglobulin variable regions, light chain primermix and the heavy chain primer set (Pharmacia) were added respectivelyto generate suitable quantities of the heavy (340 bp) and light (325 bp)chain. Following an initial 10 min dwell at 95° C., 5 U AmpliTaq GoldDNA polymerase (Applied Biosystems, Foster City, Calif.) was added. ThePCR cycle consisted of a 1 min denaturation step at 94° C., a 2 minannealing step at 55° C. and a 2 min extension step at 72° C. After 30cycles of amplification, PCR derived fragment was purified by theglassmilk beads (Bio101, Vista, Calif.) and then separated by 1.5%agarose gel electrophoresis in TAE buffer and detected by ethidiumbromide staining.

For the assembly and fill-in reaction, both purified heavy chain andlight chain fragments were added to an appropriate PCR mixturecontaining a 15 amino acid linker-primer for 8H9, dNT's, PCR buffer andAmpli Taq Gold DNA polymerase. PCR reactions were performed at 94° C.for 1 min, followed by a 4 min annealing reaction at 63° C. The heavyand light chain DNA of 8H9 were joined by the linker (GGGS)₃ (Pharmacia)into scFv in a VH-VL orientation after 7 thermocycles.

Using an assembled scFv DNA of 8H9 as template, a secondary PCRamplification (30 standard PCR cycles) was carried out using primerscontaining either Sfi I or Not I restriction sites. Thus, the Sfi I andNot I restriction sites were introduced to the 5′ end of heavy chain andthe 3′ end of light chain, respectively. Amplified ScFv DNAs werepurified by glassmilk beads and digested with Sfi I and Not Irestriction endonucleases. The purified ScFv of 8H9 was inserted intothe pHEN1 vector (kindly provided by Dr. G. Winter, Medical ResearchCouncil Centre, Cambridge, UK) containing Sfi I/ Nco I and Not Irestriction sites. Competent E.coli XL 1-Blue cells (Stratagene, LaJolla, Calif.) were transformed with the pHEN1 phagemid. Helper phageM13 K07 (Pharmacia) was added to rescue the recombinant phagemid.

2.8 Enrichment of Recombinant phagemid by Panning

50 ul of anti-8H9 idiotypic antibody 2E9 (50 ug/ml) in PBS was coated onthe 96-well PVC microtiter plates and incubated at 37° C. for 1 hour.100 ul of the supernatant from phage library was added to each well andincubated for 2 hours. The plate was washed 10 times with PBS containing0.05% BSA. Antigen-positive recombinant phage captured by theanti-idiotype MoAb 2E9 was eluted with 0.1M glycine-HCl (pH 2.2containing 0.1% BSA) and neutralized with 2M Tris solution. This panningprocedure was repeated three times. The phagemid 8HpHM9F7-1 was chosenfor the rest of the experiments.

2.9 ELISA

The selected phage was used to reinfect E.coli XL 1-Blue cells. Colonieswere grown in 2xYT medium containing ampicillin (100 ug/ml) and 1%glucose at 30° C. until the optical density of 0.5 unit at 600 nm wasobtained. Expression of scFv antibody was induced by changing to themedium containing 100 uM IPTG (Sigma-Aldrich) and incubating at 30° C.overnight. The supernatant obtained from the medium by centrifugationwas directly added to the plate coated with anti-idiotype 2E9. Thepellet was resuspended in the PBS containing 1 mM EDTA and incubated onice for 10 min. The periplasmic soluble antibody was collected bycentrifugation again and added to the plate. After a 2-hour incubationat 37° C., plates were washed and anti-MycTag antibody (clone 9E10 fromATCC) was added for 1 hour at 37° C. After washing, affinity purifiedgoat anti-mouse antibody (Jackson Immunoresearch) was allowed to reactfor 1 hour at 37° C. and the plates were developed with the substrateo-phenylenediamine (Sigma-Aldrich) as previously described.

2.10 Construction of ScFv-human-1-CH2-CH3 Mouse Human-Chimeric Gene

A single gene encoding scFv8H9 was generated by PCR method usingphagemid 8HpHM9F7-1 as the template. Secondary PCR amplification (30 PCRcycles) was carried out to insert the human IgG1 leader sequence at the5′end of the scFv8H9 DNA plus the restriction sites at the two oppositeends, i.e. Hind III and Not I, at the 5′ end of human IgG1 leader and atthe 3′ end of scFv8H9, respectively. Amplified human IgG1 leader—scFv8H9DNA was purified by glassmilk beads and digested with Hind III and Not Irestriction endonucleases according to manufacturer's instructions. TheHind III—Not I fragment of human IgG1 leader-scFv8H9 cDNA was purifiedon agarose gel and ligated into pLNCS23 vector carrying thehuman-(1-CH2-CH3 gene (kindly provided by Dr. J. Schlom, National CancerInstitute, NIH, Bethesda, Md.) (Shu et al., 1993). Competent E.coli XL1-Blue cells were transformed with pLNCS23 containing the scFv phagemid.The scFv-CH2-CH3 DNA was primed with appropriate primers and sequencedusing the Automated Nucleotide Sequencing System Model 373 (AppliedBiosystems). The sequences agreed with the cDNA sequences of the lightand heavy chains of 8H9 as well as the human-1-CH2-CH3 available fromGenBank, including the ASN 297 of the CH2 domain. In this construct,Cys220 of the genetic hinge was replaced by a proline residue, whileCys226 and Cys229 were retained in the functional hinge (Shu et al.,1993)

2.11 Cell Culture and Transfection

CHO cell or NSO myelomas cells (Lonza Biologics PLC, Bershire, UK) werecultured in RPMI 1640 (Gibco-BRL) supplemented with glutamine,penicillin, streptomycin (Gibco-BRL) and 10% fetal bovine serum(Gibco-BRL). Using effectene transfection reagent (Qiagen, Valencia,Calif.), recombinant ScFv8H9-human-1-CH2-CH3 was introduced via thepLNCS23 into CHO cell or NSO myelomas cells. Cells were fed every 3days, and G418 (1 mg/ml; Gibco-BRL) resistant clones were selected.After subcloning by limiting dilution, chimeric antibodies were producedby high density miniPERM bioreactor from Unisyn Technologies using 0.5%ULG-FBS in Hydridoma-SFM (Invitrogen Corporation, Carlsbad, Calif.). Thechimeric antibodies were purified by protein G (Pharmacia) affinitychromatography.

2.12 SDS-PAGE and Western Blot Analysis

The supernatant, the periplasmic extract and cell extract from thepositive clones were separated by reducing and nonreducing SDS-PAGE. 10%SDS-polyacrylamide slab gel and buffers were prepared according toLaemmli (Laemmli, 1970). Electrophoresis was performed at 100 V for 45min. After completion of the run, western blot was carried out asdescribed by Towbin (Towbin et al., 1979). The nitrocellulose membranewas blocked by 5% nonfat milk in TBS solution for 1 hour and incubatedwith anti-idiotype 2E9 antibody overnight at 4° C. After incubating withHRP-conjugated goat anti-rat Ig (Fisher Scientific Co., Pittsburgh,Pa.), the signal was detected by ECL system (Amersham-PharmaciaBiotech).

2.13 Cytotoxicity Assay

Target NMB7 or LAN-1 tumor cells were labeled with Na₂ ⁵¹CrO₄ (AmershamPharmacia) at 100 uCi/10⁶ cells at 37° C. for 1 hour. After the cellswere washed, loosely bound ⁵¹Cr was leaked for 1 hour at 37° C. Afterfurther washing, 5000 target cells/well were admixed with lymphocytes toa final volume of 200 μl/well. Antibody dependent cell-mediatedcytotoxicity (ADCC) was assayed in the presence of increasingconcentrations of chimeric antibody. ID complement mediated cytotoxicity(CMC), human complement (at 1:5, 1:15 and 1:45 final dilution) was usedinstead of lymphocytes. The plates were incubated at 37° C. for 4 hours.Supernatant was harvested using harvesting frames (Skatron, Lier,Norway). The released ⁵¹Cr in the supernatant was counted in a universalgamma-counter (Packard Bioscience, Meriden, Conn.). Percentage ofspecific release was calculated using the formula 100%×(experimentalcpm−background cpm)/(10% SDS releasable cpm−background cpm), where cpmwere counts per minute of ⁵¹Cr released. Total release was assessed bylysis with 10% SDS (Sigma-Aldrich), and background release was measuredin the absence of cells. The background was usually <30% of total foreither NMB7 or LAN-1 cells. Antibody 3F8 was used as the positivecontrol (Cheung et al., 1985).

2.14 Iodination

MoAb was reacted for 5 min with ¹²⁵I (NEN Life Sciences, Boston, Mass.)and chloramine T (1 mg/ml in 0.3M Phosphate buffer, pH 7.2) at roomtemperature. The reaction was terminated by adding sodium metabisulfite(1 mg/ml in 0.3M Phosphate buffer, pH 7.2) for 2 min. Free iodine wasremoved with A1GX8 resin (BioRad, Richmond, Calif.) saturated with 1%HSA (New York Blood Center Inc., New York, N.Y.) in PBS, pH 7.4.Radioactive peak was collected and radioactivity (mCi/ml) was measuredusing a radioisotope calibrator (Squibb, Princeton, N.J.). Iodineincorporation and specific activities were calculated. Trichloroaceticacid (TCA) (Fisher Scientific) precipitable activity was generally >90%.

2.15 In Vitro Immunoreactivity of Iodinated Antibody.

Immunoreactivity of radioiodine labeled antibody was assayed usingpurified anti-idiotype antibody 2E9 as the antigen. Appropriatedilutions of ¹²⁵I labeled antibodies were added to plates in duplicates,and then transferred to freshly prepared antigen plates after 1 h and 4h of binding at 4° C., respectively. The final binding step was allowedto proceed overnight at 4° C. The total percent radioactivity bound wasa summation of 3 time points for each antibody dilution. For native 8H9,maximum immunoreactivity averaged ˜65%, while 8H9 scFv-Fc chimericantibody was ˜48%.

2.16 Animal Studies

Athymic nude mice (nu/nu) were purchased from NCI, Frederick Md. Theywere xenografted subcutaneously with LAN-1 neuroblastoma cell line(2×10⁶ cells/mouse) suspended in 100 ul of Matrigel (Beckton-DickinsonBioSciences, Bedford, Mass.) on the flank. After 3 weeks, mice bearingtumors of 1-1.5 cm in longest dimension were selected. Animals wereinjected intravenously (retrorbital plexus) with 20 μCi of ¹²⁵I labeledantibody. They were anesthesized with ketamine (Fort Dodge AnimalHealth, Fort Dodge, Pa.) intraperitoneally and imaged at various timeintervals with a gamma camera (ADAC, Milpitas, Calif.) equipped withgrid collimators. Serial blood samples were collected at 5 min, 1, 2, 4,8, 18, 24, 48, 72, 120 h from mice injected with 10-11 uCi ¹²⁵I labeledantibody. Groups of mice were sacrificed at 24 h, 48 h, and 120 h andsamples of blood (cardiac sampling), heart, lung, liver, kidney, spleen,stomach, adrenal, small bowel, large bowel, spine, femur, muscle, skin,brain and tumor were weighed and radioactivity measured by a gammacounter. Results were expressed as percent injected dose per gram.Animal experiments were carried out under an IACUC approved protocol,and institutional guidelines for the proper and humane use of animals inresearch were followed.

b 3. Results

3.1 Anti-8H9-idiotypic Antibodies

Rat hybridomas specific for 8H9 and nonreactive with control murine IgG1were selected. After subcloning by limiting dilution, rat antibodieswere produced by bulk culture in roller bottles and purified by proteinG affinity column. By ELISA, 2E9, 1E12, and 1F11, all of rat subclassIgG2a, were specific for 8H9, while nonreactive with a large panel ofpurified monoclonal antibodies (Table I). In contrast, the antibodies3C2, 4C2 5C7, 7D6 and 8E12 from the same fusions were not specific for8H9. The rest of the experiments in this study was carried out usingantibody 2E9. 2E9 specifically inhibited the binding of 8H9 to LAN-1neuroblastoma (FIG. 1A) and HTB82 rhabdomyosarcoma (FIG. 1B) whilecontrol rat IgG1 (A1G4) had no effect (FIG. 1C). TABLE IAnti-8H9-idiotypic antibodies: Specificity by ELISA 1E12 1F11 3C2 4C25C7 7D6 8E12 2E9 MoAb Class λ2a λ2a λ2b μ μ λ1 μ λ2a MOPC 315 a − − +++− − − − − 20.4 λ1 − − +++ +++ ++ +++ − − 2C9 λ1 − − +++ +++ +++ +++ ++ −2E10 λ1 − − +++ − − + − − 3E6 λ1 − − +++ +++ +++ +++ +++ − 3E7 λ1 − −+++ − − + − − 4B6 λ1 − − +++ +++ ++ +++ − − 5F9 λ1 − − +++ +++ +++ +++ +− 8H9 λ1 +++ ++ +++ +++ ++ +++ − ++ MOPC 21 λ1 − − +++ +++ +++ +++ − −UJ 13A λ1 − − +++ ++ + − − − 3A5 λ2a − − +++ − − − − − HOPC-1 λ2a − −+++ + − − − − 3F8 λ3 − − +++ − − − − − FCOPC21 λ3 − − +++ ++ − ++ − −NRCO-04 λ3 − − +++ − − − − − R24 λ3 − − +++ − − − − − TIB114 λ3 − −+++ + − ++ − − Y5606 λ3 − − +++ − − − − − 3A7 μ − − + − − − − − 3G6 μ −− +++ − − − − − 5F11 μ − − + − − − − − K9 μ − − +++ − − − − − MOPC 104Eμ − − +++ − − − − −Note:OD <0.5 = −, 0.5˜1 = +, 1˜2 = ++, >2 = +++3.2 Construction and Expression of 8H9 ScFv

After three rounds of panning on 2E9, the eluted phage was used toinfect E.coli HB2151 cells and scFv expression was induced by IPTG. ScFvfrom periplasmic soluble protein fraction was tested for binding to 2E9on ELISA. Three 8H9 scFv clones when compared with the MoAb 81H9 showedsimilar titers. T he clone 8HpHM9F7-1 was selected for subcloning. TheDNA sequence of 8HpHM9F7-1 agreed with those of the 8H9VH and 8H9VL aswell as the CH2-CH3 region of human gamma chain. The supernatant,periplasmic soluble and cells pellet lysates of 8HpHM9F7-1 wereseparated by nonreducing SDS-PAGE, and analysed by western blotting. Aprotein band with molecular weight of 31 KD was found in thesupernatant, the periplasmic and cell pellet extracts using anti-MycTagantibody which recognized the sequence GAPVPDPLEPR. No such band wasdetected in control cells or 8HpHM9F7-1 cells without IPTG treatment.

3.3 Construction of Chimeric Mouse scFv-human Fc

Chimeric clones from CHO and NSO were screened by ELISA binding on 2E9.Clone 1C5 from NSO and clone IG1 from CHO were chosen for scale-upproduction. By SDS-PAGE and by western blot analysis, a single chain of54 kD under reducing conditions, and a homodimer of 102 kD undernonreducing conditions were found (FIG. 2). Antigen specificity wasdemonstrated by its binding to tumor cells (FIG. 3A, dose titration),and its inhibition by anti-idiotypic antibody 2E9 (FIG. 3B) on FACSanalysis.

3.4 In Vitro and In Vivo Properties of scFv-human Fc

The scFv-Fc chimeric antibody was inefficient in mediating ADCC in thepresence of human lymphocytes or human neutrophils (17% maximumcytotoxicity at 50:1 E:T ratio compared to >50% by the murine IgG3 MoAb3F8). It was also ineffective in CMC (data not shown). Inbiodistribution studies, it localized well to HTB82 and LAN-1 xenografts(FIG. 4). Blood clearance studies showed that chimeric 8H9 (102 kD MW)had T-1/2 of 5.3 h, and T-1/2 of 43 h when compared to averages of 4.5 hand 71 h, respectively, for native 8H9 (160 kD MW), a result of thesmaller molecular size of the construct (FIG. 5). Similarly, althoughthe percent injected dose per gram of the chimeric construct was lowerfor all tissues (average of 44% at 48 h, and 75% at 120 h), thetumor-non tumor ratios were similar to those of native 8H9 (98% at 48 hand 85% at 120 h) (Table II). TABLE II Percent Injected Dose per gramand Tumor-non-tumor ratios Percent Injected dose/gm over time (h)chimeric native Organs 24 48 120 48 120 Skin 1.4 0.7 0.2 1.8 0.7 Heart1.3 0.9 0.4 2.6 0.7 Lung 2.9 1.9 0.5 4.0 1.1 Liver 1.2 0.8 0.2 1.4 0.5Spleen 0.9 0.5 0.2 1.4 0.4 Kidney 1.5 0.9 0.5 1.9 0.5 Adrenal 0.9 0.50.5 1.8 0.3 Stomach 1.3 0.6 0.3 1.3 0.5 Small intestine 0.6 0.3 0.2 0.70.2 Large intestine 0.6 0.3 0.2 0.6 0.2 Bladder 1.2 0.6 0.4 1.0 0.6Muscle 0.5 0.3 0.2 0.5 0.2 Femur 0.6 0.3 0.2 0.8 0.2 Spine 0.6 0.4 0.20.8 0.3 Tumor 4.0 3.6 2.1 9.4 4.0 Brain 0.2 0.1 0.1 0.2 0.1 Blood 5.33.1 1.2 8.3 2.3 Tumor:Nontumor ratios over time (h) chimeric nativeOrgans 24 48 120 48 120 Skin 3.0 6.0 10.7 5.2 7.2 Heart 3.3 4.0 5.6 3.67.7 Lung 1.6 2.2 4.5 2.3 5.0 Liver 3.5 5.2 8.7 6.5 10.1 Spleen 5.1 8.112.8 6.7 15.1 Kidney 2.8 4.3 5.9 5.1 8.9 Adrenal 4.8 8.7 10.0 5.8 11.6Stomach 3.6 6.7 13.8 7.5 14.5 Small intestine 6.6 11.8 16.0 13.3 21.7Large intestine 7.1 12.7 25.9 15.7 28.5 Bladder 3.5 14.3 10.2 12.4 12.3Muscle 7.9 13.6 21.3 18.2 26.8 Femur 6.7 11.8 20.5 11.8 27.9 Spine 6.76.8 14.2 11.1 19.6 Tumor 1.0 1.0 1.0 1.0 1.0 Brain 22.7 40.9 38.7 44.668.2 Blood 0.8 1.2 1.8 1.1 2.3

4. Discussion

We demonstrated that by using rat anti-idiotypic antibody as antigensurrogate, scFv and scFv-fusion proteins can be conveniently produced.As proof of principle we utilized the anti-idiotypic antibody to clonescFv from the murine hybridoma cDNA library. The anti-idiotypic antibodywas then used to select for scFv-Fc chimeric antibodies. Both the scFvand scFv-Fc fusion protein derived by our method were specific for thenatural antigen, comparable to the native antibody 8H9. However, thescFv-Fc fusion protein could only mediate ADCC poorly and not CMC atall.

While scFv provides the building block for scFv-fusion proteins, it isnot the ideal targeting agent by itself. Being a small protein, itsclearance is rapid. Moreover, it is often retained by the kidney,delivering undesirable side effects if the scFv construct is cytotoxic.Since avidity is a key parameter in tumor targeting in vivo, its biggestlimitation is its uni-valency and often suboptimal affinity for theantigen. By using VH-VL linkers of decreasing length, spontaneousdimeric, trimeric and polymeric scFv have been produced. However, theseoligomers are not bonded by covalent linkage, and may dissociate invivo. An alternative approach is to take advantage of the human Fc,which has the natural ability to homodimerize through disulfide-bonds,thereby allowing the juxtaposition of two binding domains. Fc functionssuch as CMC and ADCC could also be achieved achieved (Shu et al., 1993;Kato et al., 1995; Brocks et al., 1997; Wang et al., 1999; Powers etal., 2001). Unlike standard 2-chain chimeric antibodies, only onepolypeptide is needed for the scFv-Fc chimeric; unbalanced synthesis ofheavy and light chains is not an issue. Larger dimeric figments are alsolikely to have increased serum-half life compared to scFv and thusimproved tumor targeting (Adams et al., 1993; Wu et al., 1996).Homodimerization of tumor cell-surface antigens by soluble antibody mayalso trigger apoptosis of tumor cells (Ghetie et al., 1997). No lessimportant is the availability of validated purification techniques usingprotein A or protein G through their binding to the Fc portion (Powerset al., 2001). Tetravalent scFv (monospecific or bispecific) are naturalextensions of the diabody approach to scFv-Fc fusion strategy (Alt etal., 1999; Santos et al., 1999), where a significant increase in aviditycan be achieved. More recently, scFv-streptavidin fusion protein hasbeen produced for pretargeted lymphoma therapy (Schultz et al., 2000).Here scFv-streptavidin forms natural tetramers, to which biotinyatedligands can bind with high affinity.

Anti-idiotypic antibodies have greatly facilitated clone selection inthe construction of soluble scFv-fusion proteins or cell bound surfacescFv. We have successfully applied similar technology to anti-GD2monoclonal antibodies (Cheung et al., 1993). Being immunoglobulins,their structure, stability, biochemistry, are generally known. Unlikenatural antigens where each individual system has its unique anddifficult to predict properties. As surrogate antigens, anti-idiotypicantibodies are ideal for standardization and quality control, especiallyfor initial clinical investigations where the nature of the antigen isnot fully understood. Potential limitations exist for the anti-idiotypeapproach Only those anti-ids (Ab2) that recognize the antigen-bindingsite of the immunizing MoAb can mimic the original antigen. A reliabletest for Ab2 is its ability to induce an antigen-specific immuneresponse. Alternatively, antigen specificity of the scFv selected by theanti-idiotype must be validated by binding to cells or membranepreparations. Once validated, the anti-idiotype can be used as antigensurrogate for cloning and assay of other scFv-fusion proteins.

Our scFv-Fc fusion protein lacks CMC and ADCC activity. This findingdiffers from previous scFv-Fc fusion proteins (Shu et al., 1993; Wang etal., 1999; Powers et al., 2001). This is unlikely to be due to the p58antigen recognized by this scFv, since anti-GD2 scFv-Fc made with thesame cassette were also deficient in CMC and ADCC activity (data notshown). One possible explanation might be due to the oligosaccharidestructures in the Fc region (Wright and Morrison, 1997). In normal IgG,these oligosaccharides are generally of complex biantennary type, withlow levels of terminal sialic acid and bisecting N-acetylglucosamine(GlcNAc), the latter being critical for ADCC. ADCC function is ofteninefficient among chimeric antibodies expressed in cell lines which lackthe enzyme.(1,4)-N-acetylglucosaminyltransferase III (GnIII) (Umana etal., 1999), that catalyzes the formation of bissecting oligosaccharides.This enzyme can be transfected into producer lines to increase the levelof bisecting GlcNAc and to increase the ADCC function of secretedchimeric antibodies (Umana et al., 1999). Since our chimeric antibodiesfrom both CHO and NSO expression systems were inefficient in CMC andADCC, both cell lines may be lacking in the GnIII enzyme. It is alsopossible that the absence of the CH1 domain in the Fc may modify theaccessability of the ASN297 residue to glycosyltransferases in somescFv-Fc constructs such as ours (Wright and Morrison, 1997). On theother hand, an scFv-Fc that lacks binding to Fc receptor may have lessnonspecific binding to white cells, thereby decreasing blood pooling intargeted therapy. These findings may have implications in scFv-Fcstrategies to improve effector functions.

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-   Wu, A. M., Chen, W., Raubitschek, A., Williams, L. E., Neumaier, M.,    Fischer, R., Hu, S. Z., Odom-Maryon, T., Wong, J. Y. and    Shively, J. E. (1996) Tumor localization of anti-CEA single-chain    Fvs: improved targeting by non-covalent dimers. Immunotechnology 2,    21-36.    Sixth Series of Experiments    Using Anti-Idiotypic Antibody to Enhance SCFV Chimeric Immune    Receptor Gene Transduction and Clonal Expansion of Human Lymphocytes

Background: Chimeric immune receptors (CIR) transduced into lymphocyteslink target recognition by single chain antibody Fv (scFv) to activationthrough CD28/TCR. signaling. The murine monoclonal antibody (MoAb) 8H9reacts with a novel antigen widely expressed on solid tumors (CancerResearch 61:4048, 2001). We want to test if its anti-idiotypic MoAb 2E9can optimize the CIR technology.

Methods: Rat anti-idiotypic MoAb 2E9 (IgG2a) was used as an antigensurrogate for initial cloning of 8H9scFv from the hybridoma cDNAlibrary. A CIR consisting of human CD8-leader sequence, 8H9scFv, CD28(transmembrane and cytoplasmic domains), and TCR-zeta chain wasconstructed, ligated into the pMSCVneo vector, and used to transfect thepackaging line GP+envAM12 bearing an amphotropic envelope.

Results: Three sequential affinity enrichments with MoAb 2E9significantly improved the percentage of producer clones positive forsurface 8H9-scFv and the efficiency of their supernatant in transducingthe indicator cell line K562. By three weeks of in vitro culture, >95%of transduced primary human lymphocytes were CIR-positive. With periodicstimulation with soluble 2E9, these lymphocytes underwent “monoclonal”expansion, reaching 50-100 fold increase by 2 months. They mediatedantigen-specific non-MHC restricted cytotoxicity efficiently. Wheninjected intravenously, they inhibited tumor growth in SCID micexenografted with rhabdomyosarcoma.

Conclusion: Anti-idiotypic antibody may provide a useful tool,especially for carbohydrate or unstable antigens, in facilitating thecloning of scFv and their CIR fusion constructs, as well as theirtransduction into human lymphocytes.

Introduction

Adoptive cell therapy using ex vivo expanded tumor-selective T-cells caneffect dramatic remissions of virally induced malignancies, a processcritically dependent on clonal frequency, where rapid exponentialexpansion of specific cytolytic T-lymphocytes (CTL) is required. T-cellsproliferate when activated (e.g. anti-CD3) but apoptose unless acostimulatory signal (e.g. anti-CD28) is provided (1). However, humantumor targets often lack costimulatory molecules (e.g. CD80), oroverstimulate inhibitory receptors (e.g. CITA) such that the CD28pathway is derailed. In addition, many tumors downregulate majorhistocompatibility complex (MHC) molecules to escape engagement by theT-cell receptor (TCR). Through genetic engineering, chimeric immunereceptors (CIR) linking tumor-selective scFv to T-cell signaltransduction molecules (e.g. TCR-zeta chain and CD28) will activatelymphocytes following tumor recognition, triggering the production ofcytokines and tumor lysis (2-7). T-cell can also be geneticallyengineered to secrete cytotoxic cytolines (8), toxins (9) or tometabolize prodrugs (10, 11). However, significant technologic gapsremain: (1) Gene transduction into human lymphocytes is inefficient, (2)antigen specific T-cells cannot be easily enriched and expanded, and (3)optimal T-cell activation may require multiple signals. Furthermore,although CIR redirected T-cells can recycle their lytic activity (12), acostimulatory signal, either through CD28 or 4-1BB engagement, may helpreduce activation-induced apoptotic death. CIR with multidomains wasrecently described, where the intracellular domain of CD28 was ligatedto the 5′ end of TCR zeta chain and introduced into Jurkat cells, withthe expected “two birds with one stone effect” when scFv binds to tumorcells (13). IL-2 production was 20 times more than CIR with zeta chainonly. Whether this same effect can be achieved with primary humanT-cells is not known.

To monitor scFv gene expression, anti-linker antibody may be useful,although its efficiency depends on the accessibility of the scFv-linkerportion. Although purified antigens can also be used to monitor scFvexpression, certain classes (complex carbohydrates or unstable antigens)can be difficult to prepare and their chemistry highly variable. Withouta standardized reagent for affinity purification or enrichment of virusproducer cells, monitoring and sorting of transduced lymphocytes, CIRtechnology remains inefficient. Recently Eshhar et al described adicistronic construct consisting of scFv-CD28-(and green fluorescentprotein (GFP), where the latter was used to monitor gene transductionand to enrich producer lines (7). Although GFP can validate the genetransfer process, its added immunogenicity and its safety in clinicalapplications remain uncertain.

Anti-idiotypic antibodies are frequently used as antigen-mimics forinfectious diseases and cancer (14, 15). Internal image ratanti-idiotypic antibodies can be conveniently produced against mouseMoAb. Since large scale production of clinical grade MoAb is nowroutine, anti-idiotypic antibodies may be ideal surrogates especially ifthe antigen is not easily available. In addition, the biochemistry ofimmunoglobulins in positive selection (panning, affinity chromatography,sorting) and binding assays is well-known and is easy to standardize. Werecently described a novel tumor antigen reactive with a murine MoAb 8H9(16). The antigen was difficult to purify given its lability andglycosylation. Here we demonstrate that anti-idiotypic MoAb can be usedas surrogate antigens for cloning CIR into lymphocytes, i.e. a CIR of8H9scFv, human CD28 and human TCR-zeta chain Anti-idiotypic MoAb allowsrapid affinity enrichment of producer cell line, monitoring of scFvexpression on cells, and in vitro clonal expansion of transducedlymphocytes. Highly cytotoxic lymphocytes, both in vitro and in vivo,can be produced in bulk. Besides providing an antigen surrogate,anti-idiotypic MoAb appears to have utility for the optimization andquality control of scFv-based gene therapies.

Materials and Methods

Materials. Cells were cultured in RPMI 1640 with 10% newbom calf serum(Hyclone, Logan, Utah) supplemented with 2 mM glutamine, 100 U/ml ofpenicillin and 100 ug/ml of streptomycin. The BALB/c myeloma proteins,MOPC-104E, TEPC-183, MOPC-351, TEPC-15, MOPC-21, UPC-10, MOPC-141,FLOPC-21, Y5606, were purchased from Sigma-Aldrich Co., St. Louis, Mo.MoAb R24, V1-R24, and K9 were gifts from Dr. A. Houghton, OKB7 and M195from Dr. D. Scheinberg, and 10-11 (anti-GM2) from Dr. P. Livingston ofMemorial Sloan-Kettering Cancer Center, New York, 528 from Dr. 3.Mendelsohn (MD Anderson Cancer Center, Houston, Tex.). 2E6 (ratanti-mouse IgG3) was obtained from hybridomas purchased from ATCC(Rockville, Md. NR-Co-04 was provided by Genetics Institute (Cambridge,Mass.). LS2D173 (anti-GM2) was provided by Dr. L. Grauer (Hybritech,Calif.). From our laboratory, 3F8 was an IgG3 MoAb specific forganglioside G_(D2) (17); 5F9, 8H9, 3A5, 3E7, 1D7, 1A7 were producedagainst human neuroblastoma, 2C9, 2E10 and 3E6 against human breastcarcinoma: 4B6 against glioblastoma multiforme. They were all purifiedby protein A or protein G (Pharmacia, Piscataway, N.J.) affinitychromatography.

Anti-8H94idiotypic MoAb. Anti-idiotypic antibodies were produced fromLOU/CN rats as previously described (18). Clones were selected based onselective binding to 5F11 antibody and not to other myelomas. Repeatedsubcloning was done using limiting dilution until the cell lines becamestable. Among the three specific rat IgG2a clones (2E9, 1E12, 1F11), 2E9was chosen for scaled up production using high density miniPERMbioreactor (Unisyn technologies, Hoplinton, Mass.), and purified byprotein G affinity chromatography (Hitrap G, Amersham-Pharmacia,Piscataway, N.J.). The IgG fraction was eluted with pH 2.7 glycine-HClbuffer and neutralized with 1 M Tris buffer pH 9. After dialysis in PBSat 4° C. for 18 hours, the purified antibody was filtered through a 0.2um Millipore filter (Millipore Inc. Bedford Mass.), and stored frozen at−70° C. Purity was determined by SDS-PAGE electrophoresis using 7.5%acrylamide gel. ELISA was used to detect rat anti-idiotypic antibodies(Ab2) as previously described (18). Rat IgG1 anti-SF11 anti-idiotypicMoAb was similarly produced.

Construction of ScFv Gene scFv was constructed from 8H9 hybridoma cDNAby recombinant phage display using a scFv construction kit according tomanufacturer's instructions with modifications (Amersham-Pharmacia).Amplified ScFv DNA was purified by glassmilk beads and digested with SfiI and Not I restriction endonucleases. The purified scFv of 8H9 wasinserted into the pHEN1 vector (kindly provided by Dr. G. Winter,Medical Research Council Centre, Cambridge, UK) containing SfiI/NcoI andNot I restriction sites. Competent E1 Coli XL 10Blue cells (Stratagene,La Jolla, Calif.) were transformed with the pHEN1 phagemid. Helper phageM13 KO7 (Pharmacia) was added to rescue the recombinant phagemid. Thephagemid 8HpHM9F7-1 was chosen for the rest of the experiments. Thesupernatant, the periplasmic extract and cell extract from the positiveclones separated by nonreducing SDS-PAGE and western blotting (19) usinganti-Myc Tag antibody demonstrated a 31 kD band.

Enrichment of Recombinant Phagemid by Panning 50 ul of anti-8H9 idiotypeantibody 2E9 (50 ug/ml) in PBS were coated on the 96-well polyvinylmicrotiter plates and incubated at 37° C. for 1 hour. 100 ul of thesupernatant from phage library were added to each well and incubated for2 hours. The plate was washed 10 times with PBS containing 0.05% BSA.Antigen-positive recombinant phage captured by the idiotype 2E9 waseluted with 0.1M HCl (pH 2.2 with solid glycine and 0.1% BSA) andneutralized with 2M Tris solution. This panning procedure was repeatedthree times.

ELISA The selected phage was used to reinfect E.coli XL 1-Blue cells.Colonies were grown in 2xYT medium containing ampicillin (100 ug/ml) and1% glucose at 30° C. until the optical density at 600 nm of 0.5 wasobtained. Expression of scFv antibody was induced by change of themedium containing 100 uM IPTG (Sigma-Aldrich) and incubating at 30° C.overnight. The supernatant obtained from the medium by centrifugationwas directly added to the plate coated with idiotype 2E9. The pellet wasresuspended in the PBS containing 1 mM EDTA and incubated on ice for 10min. The periplasmic soluble antibody was collected by centrifugationagain and added to the plate. After incubating 2 hours at 37° C., plateswere washed and anti-MycTag antibody (clone 9E10 from ATCC) was added toreact for 1 hour at 37° C. After washing, affinity purified goatanti-mouse antibody (Jackson Immunoresearch, West Grove, Pa.) wasallowed to react for 1 hour at 37° C. and the plates were developed withthe substrate o-phenylenediamine (Sigma-Aldrich).

Construction of sc8H9-hCD28_(TM)-hCD28_(cyto)-hTCRzeta-pMSCVneo Usingthe assembled gene sequences, secondary PCR amplifications usingsynthetic oligodeoxynucleotide primers (see below) were performed.Briefly, a 50 μl reaction mixture containing 200 μM of eachdeoxynucleotide triphosphate, 0.2 μM of each primer, 2 units of AmpliTagGold DNA polymerase (Applied Biosystems, Foster City, Calif.), and 50 ngof template DNA was subjected to a 10 min denaturation and activationstep at 95° C., followed by 30 cycles of denaturation (1 min at 95° C.),annealing (2 min at 55° C.), and extension (2 min at 72° C.). This wasfollowed by a final extension for 8 min at 72° C. Each of the amplifiedproducts was purified with Geneclean Kit (Bio 101, Vista, Calif.).

Synthetic Oligodeoxynucleotide Primers for DNA Amplification

hCD8a Leader—scFv—CD28:

355 S Sense Primer (Hpa I—Human CD8a Leader) 5′-TTA TTA CGA GTT/AAC ATGGCC TTA CCA GTG ACC-3′;

355 A Antisense Primer (Xho I—Human CD28 ) 5′-CTT GGT C/TCGAG TGT CAGGAG CGA TAG GCT GC-3′;

scFv8H9:

365 S Sense Primer (Cla I—8H9 heavy chain) 5′-TTA TTA CGA AT/CGAT T GCCCAG GTC AAA CTG-3′;

365 A Antisense Primer (Not I—8H9 light chain ) 5′ -CTT GGT G/CGGCCGCCTG TTT CAG CTC CAG-3′;

hTCR-zeta Chain

379S Sense primer (Bst UI—CD28 end—Xho I—hTCR zeta [cytoplasmic domain])5′-CG/C GAC TTA GCA GCC TAT CGC TCC TGg CAC/TCG AGa AGA GTG AAG TTC-3′;

379A Antisense Primer (BglII—hTCR z) 5′-CTT GGT A/GA TCT TCA GCG AGG GGGCAG GGC-3′.

Templates for DNA Amplification and Construction The single geneencoding hCD8a-leader-sc3G6-CD28 was previously described (20). Its cDNAwas generated by PCR using the Hpa I, Xho I fragment ofhCD8a-leader-scFv-CD28 cDNA, and ligated into pMSCVneo vector (Clontech,Palo Alto, Calif.). ScFv-8H9 was amplified from the 8HpHM9F7-1 phagemid.Excised 8H9 scFv gene was then swapped into thehCD8a-leader-scFv3G6-CD28 cassette of pMSCVneo using the Cla I—Not Irestriction enzymes. Human TCR-zeta-chain was amplified from the plasmidpcDNA3.1/VJABLZH (kindly provided by Dr. Ira Bergman, University ofPittsburgh, Pa.), and ligated downstream of CD28 gene, using Xho I andBgl II restriction sites. Using the method supplied by manufacturer(Stratagene), competent E.coli XL 1-Blue cells were transformed with thevector pMSCVneo containing the insert. All gene constructs were checkedby DNA sequencing.

Cell Culture and Transfection The amphotropic packaging cell lineGP+envAM12 and all retroviral producer lines were maintained inDulbecco's modified Eagle's medium (Gibco-BRL, Gaithersburg, Md.)supplemented with glutamine, penicillin, surepeomycm (Gibco-BRL), and10% fetal bovine serum (Gibco-BRL). Using effectene transfection Reagent(Qiagen, Valencia, Calif.), recombinant retrovirus was produced by thetransfection of vector DNA into GP+envAM12 packaging cells (kindlyprovided by Genetix Pharmaceuticals, Cambridge, Mass.). Cells were fedevery 3 days with G418 (400 ug/ml; Gibco-BRL). Resistant clones wereselected after a 10-day period.

Enrichment and Cloning of Packing Lines by Affinity Column Theretroviral producer lines were affinity enriched using MACS goatanti-rat IgG MicroBeads on the MiniMACS system (Miltenyi, Auburn,Calif.). In brief, the transduced packing lines were reacted withpurified rat anti-idiotypic antibodies (10 ug per 10⁶ packing cells) onice for 30 minutes, washed and then applied to the anti-rat column. Cellwere eluted according to manufacturer's instructions and recultured at37° C. for 24 hours. Following staining with anti-idiotypic antibody 2E9or 1E12, immunofluorescence was detected with FITC conjugated mouseanti-rat IgG antibody and analyzed by a FACSCalibur flow cytometer(Becton Dickinson Immunocytometry systems, San Jose, Calif.). A seriesof three affinity purifications is performed on the retroviral producerline before subcloning by limiting dilution. Virus-containingsupernatant from each clone was used to infect K562 cells, and genetransduction was measured by surface expression of scFv on K562 usingFACS. One of the scFv-transduced K562 cell lines was further enriched byMACS system before cloning by limiting dilution.

Peripheral Blood Mononuclear cells (PBMCs) PBMCs were isolated bycentrifugation on Ficoll (density, 1.077 g/ml) for 30 min at 25° C. andwashed twice with PBS. They were activated with soluble anti-CD3 (1μg/ml; clone OKT3; PharMingen, San Diego, Calif.) and anti-CD28 (1ug/ml; clone CD28.2; PharMingen) MoAbs for 3 days at 37° C. In someexperiments, immobilized anti-CD3 and anti-CD28 MoAbs were used, where12-well non-tissue culture-treated plates were incubated with theantibody (1 μg/ml in PBS) at 1 ml/well for 4 hours at 37° C. The coatedplates were blocked with 1% HSA in PBS for 30 min at room temperature,washed once with PBS, and then used for PBMC activation. PBMCs (10⁶/ml)were cultured in RPMI 1640 supplemented with 10% human AB serum (GeminiBio-Products, Woodland, Calif.), 50 μM 2-mercaptoethanol, 2 μML-glutamine, and 1% penicillin-streptomycin (Gibco-BRL), for a total of3 days before retroviral transfection.

Retroviral Transduction Protocol The target cells ( e.g. K562 orcultured PBMCs) were resuspended at a concentration of 1-5×10⁵ cells/mlof freshly harvested supernatant from retroviral producer cells,containing 8-10 ug/ml hexadimethrine bromide (polybrene, Sigma),centrifuged at 1000×g at room temperature for 60 minutes, and thencultured in 12-well tissue culture plates overnight. The viralsupernatant was then aspirated and fresh IMDM (Gibco) medium containing100 U/ml of IL2 and changed approximately every 5 days to maintain acell count between 1-2×10⁶ cells/ml (21). After 2 weeks in culture,soluble anti-idiotypic antibody 2E9 was added at 3-10 ug/ml to thetransfected lymphocytes for 3 days out of every 2-week culture period,to ensure clonal expansion of the scFv-positive transfected lymphocytes.

Cytotoxicity Assay Neuroblastoma targets NMB-7 and LAN-1 orrhabdomyosarcoma HTB-82 tumor cells were labeled with Na₂ ⁵¹CrO₄(Amersham Pharmacia Biotechnology Inc., Piscataway, N.J.) at 100 uCi/10⁶cells at 37° C. for 1 hour. After the cells were washed, loosely bound⁵¹Cr was removed by washing. 5000 target cells/well were admixed withlymphocytes to a final volume of 200 μl/well. Following a 3 minutecentrifugation at 200×g, the plates were incubated at 37° C. for 4hours. Supernatant was harvested using harvesting frames (Skatron, Lier,Norway). The released ⁵¹Cr in the supernatant was counted in a universalgamma-counter (Packard Bioscience, Meriden, Conn.). Percentage ofspecific release was calculated using the formula 100%×(experimentalcpm−background cpm)/(10% SDS releasable cpm−background cpm), where cpmare counts per minute of ⁵¹Cr released. Total release was assessed bylysis with 1 0% SDS (Sigma-Aldrich), and background release was measuredin the absence of cells. The background was usually <30% of total forthese cell lines.

Mice and Treatment CB-17 SCID-Beige mice were purchased from Taconic(Germantown, N.Y.). Tumor cells were planted (2×10⁶ cells) in 100 ul ofMatrigel (BD BioSciences, Bedford, Mass.) subcutaneously. Followingimplantation, tumor sizes (maximal orthogonal diameters) were measured.Tumor volume was calculated as 4Br^(3/)3 where r is the mean tumorradius. Treatment studies started in groups of 5 mice per cage whentumor diameter reached 0.8 cm, usually by one week of tumorimplantation. Mice received 5 weekly intravenous lymphocyte injectionsby retroorbital route, 2×10⁶ per injection together with 500 U of IL-2ip. 50 ug of anti-idiotypic antibody was administered ip 3 days aftereach lymphocyte injection. Tumor sizes were measured twice a week.Experiments were carried out under an IACUC approved protocol andinstitutional guidelines for the proper, and humane use of animals inresearch were followed.

Statistical Analysis Tumor growth was calculated by fitting a regressionslope for each individual mouse to log transformed values of tumor size.Mean slope scores were back-transformed to give an estimate of thepercent increase in tumor size per day. Slopes were compared betweengroups.

Results

Anti-8H9-idiotypic antibodies Rat hybridomas specific for 8H9 andnonreactive with control murine MoAb (IgM, IgG1 and other subclasses)were selected. By ELISA, 2E9, 1E12, and 1F11 were all of rat subclassIgG2a. The antibody 2E9 was chosen for the rest of the experiments.

Construction and expression of 8H9 scFv After secondary PCRamplification, the PCR product of scFv fitted with Sfi I and Not Irestriction sites were inserted into pHEN1 vectors. Three rounds ofpanning were conducted to enrich for 2E9-binding recombinant phages. Thephages eluted from the third round panning were used to infect E.coliHB2151 cells and induced by IPTG for expression. ScFv periplasmicsoluble protein was allowed to react in plates coated with 2.5 ug2E9/well and assayed by ELISA as described in Material and Methods. Theclone 8HpHM9F7-1 was selected for subcloning. The scFv DNA sequence of8HpHM9F7-1 agreed with those of the VH and VL regions of the MoAb 8H9.The supernatant, periplasmic soluble and cells pellet lysates of8HpHM9F7-1 were separated by nonreducing SDS-PAGE, and analyzed bywestern blotting. A protein band with the apparent molecular weight of31KD was found in the supernatant, the periplasmic and cell pelletextracts using anti-MycTag antibody which recognized the sequenceGAPVPDPLEPR. No such band was detected in control cells or 8HpHM9F7-1cells without IPTG treatment.

Construction of sc81H9-CD28-hTCRzeta-pMSCVneo Using the assembled genesequences, secondary PCR amplifications using syntheticoligodeoxynucleotide primers were performed using syntheticoligodeoxynucleotide primers 355S, 355A for the hCD8a leader—scFv—CD28 ,365S, 365A for scFv8H9, and 379S, 379A for hTCR-zeta chain. The finalgene construction hCD8_leader-8H9scFv-hCD28_(TM)-hCD28_(cyto)-TCR wastransfected into the amphotropic packaging line GP+envAM12, and selectedin G418.

Enrichment and cloning of packing lines by affinity column Theretroviral producer lines were affinity-enriched using MACS goatanti-rat IgG MicroBeads on the MiniMACS system. Following eachenrichment, viral supernatant from the producer line was used to infectthe erythroleukemia line K562. Surface 8H9-scFv expression on both theproducer lines and the transfected K562 (3-5 days after infection) weremeasured by immunofluorescence using anti-idiotypic antibody 2E9. Witheach successive affinity enrichment (FIGS. 1A and 1C) of producer lineand subsequent successive subcloning (FIGS. 1B and 1D), the surfaceexpression (mean fluorescence) of 8H9-scFv increased and became morehomogeneous for the producer clones (FIGS. 1A and 1B) and for theindicator line K562 FIGS. 1C and 1D).

Retroviral transduction of primary human peripheral blood mononuclearcells Following activation in vitro with soluble anti-CD3 and anti-CD28,primary human peripheral blood mononuclear cells were infected with thevirus from producer line supernatant by centrifugation at 1000×g for 60minutes at room temperature. By 21 days of in vitro culture, close to100% of cells were scFv-positive by FACS (FIG. 2). This clonal evolutionto homogeneity was found in CD4+, CD8+ and the small CD56+ populations.Soluble anti-idiotypic MoAb 2E9 was added at 3-10 ug/ml to thetransfected lymphocytes for 3 days out of every 2 weeks, to stimulateclonal expansion of the scFv-positive transfected lymphocytes (FIG. 3).ScFv expression was constant throughout until at least day 62 (FIG. 2),while the cells underwent active clonal expansion of 100-fold. Theproportion of CD8+ cells increased steadily from an initial 20-60% to90% by day 40 of culture.

Transduced lymphocytes carried out efficient non MHC-restrictedcytotoxicity in vitro against neuroblastoma and rhabdomyosarcoma Invitro cytotoxicity against NMB-7 (FIG. 4A) and LAN-1 (FIG. 4B)neuroblastoma, or rhabdomyosarcoma HTB-82 (FIG. 4C) were efficient, allinhibitable by 8H9 antibody demonstrating antigen specificity. Daudicell line (FIG. 4D) was not killed because it was antigen-negative. Thiscytotoxicity was independent of target HLA expression or HLA types.Unmodified lymphocytes from the same donor, cultured under the sameconditions (100 U/ml of IL2), did not show antigen-specific killing(LAK, FIG. 4).

Inhibition of rhabdomyosarcoma tumor xenografted in SCID mice. Humanrhabdomyosarcoma was strongly reactive with 8H9, but not with 5F11(anti-GD2) antibodies. To study the in vivo effects of 8H9scFv-CIRgene-modified lymphocytes, we used 5F11 scFv-CIR as control.5F11scFv-CIR modified lymphocytes could kill tumors in vitro, but onlyif they were GD2-positive (data not shown). When subcutaneous tumorimplants grew to 0.8 cm diameter, mice were treated with 2×10⁶gene-modified human lymphocytes intravenously plus 500 U of IL2intraperitoneally once a week for a total of 5 weeks. 50 ug ofanti-idiotypic antibody 2E9 was given ip 3 days after each lymphocyteinfusion. All groups received IL2. Control groups received either nocells+2E9, cultured unmodified lymphocytes+2E9 (LAK), or 5F11scFv-CIRmodified lymphocytes+anti-idiotype 1G8 (specific for 5F11 idiotype).Suppression of tumor growth was most significant with lymphocytestransduced with the 8H9scFv-CIR gene (p=0.066, FIG. 5). Although5F11scFv-CIR modified lymphocytes also delayed tumor growth, they werenot different from unmodified lymphocytes.

Discussion

The use of retroviral vectors to transduce chimeric immune receptorsinto primary human lymphocytes has been limited by the low gene transferefficiency when viral supernatant infections were carried out Transferrates into primary human T cells using amphotropic virus ranged from 1to 12% (22). Several strategies were explored to increase thetransduction rates to 20-50%. These include: (1) using gibbon apeleukemia virus (GaLV strain SEATO) pseudotyped virions (20, 23, 24), (2)coculturing producer and target cells (25) where the clinical safety wasof some concern, (3) using phosphate depletion followed bycentrifugation and incubation at 32° C. (22), (4) adding fibronectinCH296 to enhance virus/lymphocyte interactions (26). More recently,Eshhar et al described a dicistronic construct consisting ofscFv-CD28-(and green fluorescent protein (GFP), where the latter wasused to monitor gene transduction and to enrich producer line (7). Inour study, we used anti-idiotypic antibody to select for high surfacescFv-expressing producer lines with improved efficiency of genetransduction. More importantly, lymphocytes transduced by CD-28−.chimeric fusion receptors proliferated in the presence of theanti-idiotypic MoAb to become “monoclonal” with respect to scFvexpression, in both the CD4+ and CD8+ populations. These lymphocytespossessed antigen-specific tumorcidal activity both in vitro and in vivothat was non-MHC restricted. Whether CD56-positive cells (presumably NKcells) acquire similar abilities will need further studies, althoughactivation of NK cells through CD28 signaling has been reportedpreviously (27).

We have shown that anti-idiotypic antibodies can facilitate cloneselection in the construction of soluble scFv-fusion proteins or cellbound surface scFv. We have successfully applied similar technology tothe GD2 antigen system (unpublished data). Being immunoglobulins, theirstructure, stability, biochemistry are generally known. This is incontrast to natural antigens where each individual system has its uniqueand often difficult-to-predict properties. As surrogate antigens,anti-idiotypic MoAb are ideal for standardization and quality control,especially for initial clinical investigations of carbohydrate antigensor when the nature of the antigen is not fully understood.

The advantage of using anti-idiotypic antibody for affinity purificationand for clonal expansion of gene-modified lymphocytes are many fold. Toprepare polyclonal CTLs specific for a tumor target, lymphocytes have tobe pulsed periodically in vitro with the tumor cells(21). Clearly thiscan create safety (tumor contamination) and quality control issues. Incontrast, anti-idiotypic MoAb can be manufactured under standard goodmanufacturing practice (GMP) conditions, with ease of manipulation bothin vitro or in vivo. Another advantage of anti-idiotypic MoAb is itsability to mark the clonal population of target-specific lymphocytes.Although tetramers can mark TCR and T-cell clones, identity of thepeptide antigen is required and this technology is not easily available.Furthermore, anti-idiotypic MoAb can mark T-cell clones in vivo whenradiolabeled, an option not yet possible with tetramers. Finally, thepotential of anti-idiotypic MoAb to activate the transduced lymphocytesin vivo is appealing, especially when tumor cells are poorlyimmunogenic, or when they are scarcely distributed. Although we usedanti-idiotypic MoAb in our SCID mice experiments, this strategy clearlyrequires further optimization after a better understanding of in vivobiology of these transduced cells become available.

Despite these encouraging results, other structural issues of CIRtechnology will have to be considered for future optimization. Thechoice of the appropriate spacer (between scFv and signaling molecule),transmembrane domain and the signaling molecules may be important (28).That 8H9scFv-modified T-cells proliferate with anti-idiotype and killantigen-positive tumor cells argue strongly that the CD28 trans-membranedomain in this CIR design does not require a CD8 hinge, permittingeffective interaction with soluble as well as cell-bound antigens. Thisinteraction effects positive lymphocyte signaling, for both survival andactivation, as previously reported for similar chimeric fusion proteincontaining both CD28 and TCR-chains (13). It is possible that the levelof activation could be improved by the addition of a hinge or theadoption of other trans-membrane domains, as previously suggested (29).Previous reports have suggested that a human IgG hinge-CH2-Ch3 spacercan optimize T-cell activity, surface expression, and target affinity(28, 30). Moreover, using domains or molecules further downstream in theT-cell activation pathway could potentially overcome the T-cell defectscommonly found in cancer patients (31). Another variable in T-cellactivation is the affinity of interaction between TCR and MHC peptidecomplex (32). Whether a chimeric receptor of low affinity scFv maybetter mimic naive TCR interaction needs to be further tested. Anoptimal density of CIR for T-cell activation is probably important (33),since excessive TCR signaling may trigger premature death. In addition,since most target antigens are not tumor-specific, it may be useful tostandardize the level of expression of CIR such that an engineeredT-cell is optimally activated only by a narrow threshold of antigen.

The choice of tumor system and antigen target will likely determine theclinical success of CIR strategy. Primary lymphoid tumors e.g. B-celllymphomas have distinct attributes. Because of their innate tropism,T-cells home to these lymphomas. In addition, these tumors have uniquetumor antigens with homogeneous expression that do not modulate from thecell surface (e.g. CD20). Furthermore, these B-cell tumors expresscostimulatory molecules (30). Most solid tumors lack these attributes.However, metastatic cancers in lymph nodes, blood and bone marrow areunique compartments where CIR technology may be applicable. Depending onthe compartment, targeting of T-cells may require different chemokinereceptors or adhesion molecules. For example, while L-selectin isrequired for homing to lymphoid organs, its role for trafficking toother metastatic organs such as marrow is less well defined.

In adoptive cell therapies, the precise evaluation of the quantity andpersistence of these cells in vivo, as well as their distribution andfunction within tissues is critical (34). In studies of T-cell therapy,this is of particular importance since many infused cells will undergoactivation-induced death in vivo (35), or immune elimination ofgene-modified cells may occur, especially following repeated injections(36). The development of sensitive, accurate and reproducible methods toquantify gene-marked cells in peripheral blood and tissues are essentialfor defining the long-term fate of adoptively-transferred cells. WhilePCR and quantitative RT-PCR methods are ideal for studying tissuesextracts, anti-idiotypic MoAb will provide useful tools to enumerateindividual scFv-positive cells in blood, marrow and tumor. In addition,noninvasive imaging methods using radiolabeled anti-idiotypic MoAb mayalso be possible. Similar to the marker gene HSV-tk that allows cells tobe tracked and quantified by the substrate ¹³¹I-FIAU or ¹²⁴I-FRAU,anti-idiotypic MoAb labeled with either “¹³¹I or ¹²⁴I can also takeadvantage of instrumentation and software developed for SPECT andPET/micro-PET imaging, respectively. These tools can provideunprecedented precision and dynamic information on cell traffic inpatient trials.

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Metastatic rhabdomyosarcoma is a chemotherapy-responsive tumor. However,cure is elusive because of the failure to eradicate minimal residualdisease (MRD). MoAb may have potential for selective targeting oftherapy to MRD. Few MoAb of clinical utility have been described forRMS. We previously reported the broad tumor reactivity of a murine MoAb8H9 with low/no staining of normal human tissues. The target antigen wastypically expressed in a homogeneous fashion among neuroectodermal(neuroblastoma, Ewing's sarcoma, PNET, brain tumors), mesenchymal (RMS,osteosarcoma, DSRT, STS) and select epithelial tumors. Of 25 RMS tumors,24 stained positive. Radioimmunolocalization of subcutaneous RMSxenografts in SCID mice was studied using radiolabeled 8 H9. Followingiv injection of 120 u Ci of ¹²⁵I-8H9, selective tumor uptake was evidentat 4 to 172 hrs after injection, with a blood TY₂ of 0.8 h and T1/2 of26 h. Mean tumor/tissue ratios were optimal at 172 h (for lung 4, kidney7, liver 9, spleen 10, femur 16, muscle 21, brain 45). Averagetumor/blood ratio were 0.7, 1.4 and 1.6, and tumor uptake was 9.5±3.4,13.3±1.5, and 5.3±0.9% injected dose per gin at 24, 48 and 172 h,respectively. The selective targeting of 8H9 to RMS xenografts suggestsits potential for radioimmunodetection and MoAb-based targeted therapyof MRD in RMS.

Radioimmunotargeting of Human Rhabdomyosarcoma Using Monoclonal Antibody8H9

Abstract

Purpose: Although metastatic rhabdomyosarcoma (RMS) is chemotherapy andradiotherapy—responsive, few patients are cured. 8H9, a murine IgG₁monoclonal antibody (MoAb), recognizes a unique cell surface antigenthat has restricted expression on normal tissues but is broadlydistributed on neuroectodermal, epithelial and mesenchymal tumorsincluding RMS. In this report we test its immunotargeting potential inmice with subcutaneous human RMS.

Experimental design: Athymic nude mice with established RMS xenograftswere injected intravenously with ¹²⁵I-8H9 or ¹²⁵I-control MoAb. ¹²⁵I-8H9immunoreactivity was tested on solid-phase anti-8H9-idiotypic rat MoAb2E9. Mice were imaged using a gamma camera and biodistribution ofradiolabeled antibodies determined. The anti-tumor effect was studiedfollowing intravenous (IV) administration of 18.5 MBq ¹³¹I-8H9.

Results: Following IV injection of 4.44 MBq of ¹²⁵I-8H9, selective tumoruptake was evident 4 to 172 h after injection. Average tumor uptake was11.5±3.9, 15.1±3.7, and 5.4±1.2% injected dose per gm at 24, 48 and 172h, respectively. Mean tumor/tissue ratios were optimal at 172 h (forlung, 4, kidney 6, liver 7, spleen 11, femur 14, muscle 18, brain 48).Tumor/tissue ratios were improved when a lower dose (0.74 MBq) of¹²⁵I-8H9 was injected. No hematological or histological abnormalitieswere observed. Mice injected with ¹²⁵I-negative control did notdemonstrate specific tumor uptake. In contrast to ¹³¹I-control treatedmice, which showed unabated tumor progression, mice treated with 18.5MBq of ¹³¹I-8H9 showed tumor suppression of >50%.

Conclusions: Radiolabeled 8H9 effectively targeted RMS xenografts andmay have a potential clinical role in immunodetection and immunotherapy.

Introduction

Metastatic rhabdomyosarcoma (RMS) is associated with a dismal prognosiswith reported cure rates of no greater than 25% despite demonstratedchemosensitivity and radiosensitivity (1,2,3). Myeloablativechemotherapy with autologous stem cell rescue has failed to impactsurvival (4,5). The failure to eradicate minimal residual disease (MRD)leads to local and distant relapses for both alveolar and embryonal RMS.Alternative strategies to target MRD are therefore warranted. Monoclonalantibodies (MoAbs) have recently been reported to be of clinical benefitin the treatment of solid tumors. In children with high-riskneuroblastoma (NB), the addition of the anti-ganglioside G_(D2) antibody3F8 to a multimodality approach has significantly improved prognosis (6)without increasing long-term toxicity (7). Radiolabeled antibodies canselectively deliver radiation to human tumors. Demonstration of specificbinding to NB xenografts by ¹³¹I-3F8 was initially demonstrated inxenograft models (8). Indeed, ¹³¹I-3F8 completely ablated NB xenograftsin athymic nude mice with reversible toxicity (9). Based on thepharmacokinetics and dosimetry calculations to tumors and normal tissuesradioimmunodetection and radioimmunotherapy, clinical protocolsutilizing ¹³¹I-3F8 were initiated in patients with NB. Subsequently,effective and specific targeting of NB in humans was demonstrated(10,11), and later utilized both for detection and therapy.

The adoption of a similar strategy to RMS has been limited by thepaucity of antigens that can be targeted by MoAbs. Most antigensexpressed on RMS either have a nuclear or cytoplasmic localization whichmakes them inaccessible to MoAbs, or are coexpressed on normal tissuesthus limiting their clinical utility (Table 1). The PAX-FKHR fusiontranscript is specific for alveolar RMS. It has been used in thedetection of micrometastases in alveolar RMS by RT-PCR (12, 13) and as atumor antigen for the generation of cytotoxic T-cells (14). However, itsnuclear localization shields the intact protein from antibody-basedtargeting approaches. Furthermore, for the more frequent embryonalvariant, such specific markers are not yet available. We recentlydescribed a novel tumor antigen with an apparent molecular weight of 58kD (15) recognized by the MoAb 8H9. This glycoprotein is expressed oncell surface of a broad spectrum of solid tumors in childhood andadults, including both alveolar and embryonal RMS and has restricteddistribution on normal tissues. We now report the in vivo targeting of¹²⁵I and ¹³¹I labeled 8H9 in human RMS xenografts. TABLE 1 Previouslyreported antigens on rhabdomyosarcoma Antigen LocalizationCrossreactivity Desmin (22) cytoplasm Skeletal, Cardiac and SmoothMuscle Cytokeratin (23) cytoplasm Epithelial cells EMA (23) cytoplasmEpithelial cells Vimentin (23) cytoplasm All mesenchymal tissues NSE(23) cytoplasm Brain and neural tissue MYOD1 (17) nucleus Restricted toRMS Ag BW575 (18) cell membrane Neural cells Myosin (19) cell membraneMuscle cells 5.1 H11 (25) cytoplasm Neural cells IGFI receptor (21) cellmembrane Normal cells Fetal acetylcholine cell membrane Extraocularmuscles, receptor (20) thymus, denervated skeletal muscle

TABLE 2 % injected dose/gram of ¹²⁵I-8H9 distributed in HTB82 xenograftsand normal tissues 24, 48 and 172 hours after injection 24 h 48 h 172 h(n = 9 mice) (n = 9 mice) (n = 8 mice) Mean ± SD Mean ± SD Mean ± SDAdrenal 1.4 ± 1.6 1.4 ± 0.5 0.4 ± 0.3 15 Bladder 2.6 ± 1.2 2.9 ± 0.8 0.9± 0.6 Blood 14.1 ± 3.0  10.7 ± 2.1  3.2 ± 0.9 Brain 0.3 ± 0.1 0.3 ± 0.10.1 ± 0.0 Femur 1.4 ± 0.5 1.1 ± 0.5 0.4 ± 0.1 Heart 4.3 ± 1.9 2.9 ± 0.50.9 ± 0.2 Kidney 3.9 ± 1.6 3.0 ± 0.7 0.8 ± 0.3 Large Intestine 1.7 ± 0.61.2 ± 0.3 0.2 ± 0.1 Liver 4.0 ± 1.7 2.2 ± 0.3 0.7 ± 0.3 Lung 5.7 ± 3.55.3 ± 1.1 1.4 ± 0.5 Muscle 1.2 ± 0.6 1.1 ± 0.4 0.3 ± 0.1 20 Skin 2.3 ±1.6 2.5 ± 1.5 0.6 ± 0.4 Small Intestine 1.5 ± 0.4 1.1 ± 0.2 0.3 ± 0.1Spine 2.1 ± 0.8 1.7 ± 0.7 0.5 ± 0.2 Spleen 5.8 ± 2.4 3.3 ± 0.8 0.5 ± 0.2Stomach 2.4 ± 2.1 1.6 ± 0.7 0.5 ± 0.4 Tumor 11.5 ± 3.9  15.1 ± 3.7  5.4± 1.2

TABLE 3 Tumor:non-tumor ratios in mice injected with 0.74 MBq comparedto 4.44 MBq of ¹²⁵I-8H9 172 h post injection (5 mice per group) 0.74 MBq4.44 MBq Mean ± SD Mean ± SD Adrenal 26.3 ± 20.4 12.5 ± 3.6 Bladder 35.0± 31.4  7.9 ± 1.5 Blood 2.6 ± 1.7  1.7 ± 0.3 Brain 150.9 ± 36.1   51.9 ±20.1 Femur 26.7 ± 20.6 13.7 ± 2.0 Heart 11.5 ± 8.5   5.7 ± 1.0 Kidney8.4 ± 3.5  6.5 ± 1.4 Large Intestine 32.3 ± 18.6 23.0 ± 5.0 Liver 13.0 ±6.7   7.0 ± 0.9 Lung 7.7 ± 6.0  4.1 ± 0.6 Muscle 33.0 ± 22.3 18.9 ± 4.4Skin 13.0 ± 8.9   7.2 ± 3.1 Small Intestine 29.4 ± 17.0 20.8 ± 6.4 Spine20.4 ± 11.1 10.3 ± 3.7 Spleen 16.4 ± 11.3 11.9 ± 2.0 Stomach 23.4 ± 15.914.5 ± 4.3 Tumor 1.0 ± 0   1.0 ± 0 

TABLE 4 Biodistribution of ¹²⁵I-8H9 and ¹²⁵I-2C9 in mice with HTB82xenografts 120 h after injection (values represent % injecteddose/gram). ¹²⁵I-8H9 ¹²⁵I-2C9 Mean ± SD Mean ± SD Adrenal 0.5 ± 0.2 0.7± 0.4 Bladder 1.5 ± 0.8 1.5 ± 0.4 Blood 4.6 ± 0.7 8.4 ± 1.4 Brain 0.1 ±0.1 0.2 ± 0.1 Femur 0.6 ± 0.1 0.9 ± 0.2 Heart 1.0 ± 0.2 1.7 ± 0.4 Kidney1.2 ± 0.3 1.4 ± 0.4 Large intestine 0.4 ± 0.3 0.5 ± 0.1 Liver 0.9 ± 0.11.4 ± 0.1 Lung 2.9 ± 0.7 5.3 ± 1.5 Muscle 0.4 ± 0.1 0.5 ± 0.1 Skin 0.8 ±0.1 1.0 ± 0.3 Skin 0.8 ± 0.1 1.0 ± 0.3 Small intestine 0.4 ± 0.1 0.6 ±0.1 Spine 0.6 ± 0.1 1.3 ± 0.5 Spleen 1.3 ± 0.6 2.2 ± 0.5 Stomach 0.5 ±0.2 1.1 ± 0.2 Stomach contents 0.3 ± 0.1 0.3 ± 0.2 Tumor 7.2 ± 0.9 2.5 ±0.9

TABLE 5 Mean hematological and liver function parameters in mice (5 pergroup) injected with ¹³¹I-8H9 Reported Day 15 Day 30 normal values CBCHb (g/dl)  11.2 ± 0.3   13.1 ± 3.2  11.0-14.0 WBC (10³)  4.43 ± 0.7  6.2 ± 2.7  2.8-9.2 Platelets (10³)  1309 ± 371   1300 ± 798  1523 ±218  Segmented (%)  46.8 ± 9.9   42.5 ± 11.4   42-45.5 Lymphocytes (%) 49.6 ± 11.6  51.2 ± 16.2 54.5-58   Liver function tests (pooled serum)Alk. Phosphatase (IU/L) 96 174  66-258 ALT (IU/L) 36 33  62-121 AST(IU/L) 169 93  87-318 GGT (IU/L) 0 0 Albumin (g/dl) 3.1 4.8 2.5-4.8Total protein (g/dl) 5.5 5.1 3.5-7.2 Total bilirubin (mg/dl) 0.1 0.30.1-0.9Materials and MethodsMonoclonal Antibodies

MoAb 8H9 The m urine MoAb 8H9 was produced by hyperimmunizing BALB/cmice with human neuroblastoma as previously described. (15).

MoAb 2C9 Using similar methods, mice were immunized with human breastcancer and the hybridoma demonstrating specificity against cytokeratin 8was isolated.

Anti-idiotypic MoAbs Rat anti-8H9-idiotype MoAbs were produced byimmunizing LOU/CN rats with purified 8H9. Following in vitrohybridization with the myelomas SP2/0 or 8653, three IgG₂a clones (2E9,1E12 and 1F11) were selected for their high binding and specificity byELISA. When tested against a panel of 23 other myelomas, nocrossreactivity was found. T he anti-idiotypic hybridomas were clonedand the antibody 2E9 chosen for scaled up production using high-densityMiniPERM bioreactor (Unisyn technologies, Hopkinton, Mass.).Anti-idiotypic antibodies were further purified by protein G affinity(Hitrap G, Pharmacia, Piscataway, N.J.) chromatography and filteredthrough a 0.2 μm Millipore filter (Millipore Inc., Bedford, Mass.).

Cell Lines

RMS cell line HTB82 and small cell lung cancer cell line HTB119 (8H9negative control) were purchased from American Type Culture Collection,Bethesda, Md. Cell lines were grown in RPMI (Gibco BRL, Gaithersburg,Md. supplemented with 10% newborn calf serum (Hyclone, Logan, Pa.), 2 mMglutamine, 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco-BRL,Gaithersburg, Md. Cells were cultured in a 37° C. incubator andharvested using 2 mM EDTA.

Iodination

MoAb 8H9 was allowed to react for 5 min with ¹²⁵I or ¹³¹I (NEN LifeSciences, Boston, Mass.) and chloramine T (1 mg/ml in 0.3M Phosphatebuffer, pH 7.2) at room temperature. The reaction was stopped by addingsodium metabisulfite (1 mg/ml in 0.3M Phosphate buffer, pH 7.2) for 2min. Radiolabeled MoAb was separated from free iodine using A1GX8 resincolumn (BioRad, Richmond, Calif.) saturated with 1% HSA (New York BloodCenter Inc., Melville Biologics Division, New York, N.Y.) in PBS, pH7.4. Peak radioactive fractions were pooled and the radioactivity(MBq/ml) was measured using a radioisotope calibrator (Squibb,Princeton, N.J.). Iodine incorporation and specific activities werecalculated. Trichloroacetic acid (TCA) (Fisher Scientific, Pittsburgh,Pa.) precipitation was used to assess the percentage of protein bound¹²⁵I or ¹³¹I. Thin layer chromatography was performed by running 1 μl of125I-8H9 on a silica gel on glass TLC plate (Sigma Chemical, St. Louis,Mo.) and scanning it with System 200 Imaging Scanner (Bioscan,Washington, DC).

In Vitro Immunoreactivity of Iodinated 8H9

Immunoreactivity of labeled antibody was determined by a specificmicrotiter solid phase radioimmunoassay developed using theanti-8H9-idiotypic antibody 2E9 as the antigen. Briefly, microtiterplates were precoated with diminishing concentrations of 2E9.Appropriate dilutions of ¹²⁵I-8H9 were added in duplicate. Binding wasmaximized by serial incubations at 4° C in 3 separate antigen plates forperiods of 1 h, 4 h and overnight respectively. The percent of boundactivity was summed for each dilution to obtain the maximum percentbinding. Similar assay was carried out to assess immunoreactivity of¹³¹I-8H9.

Immunoreactivity was also measured by specific binding to cell pellets.HTB82 cells were suspended in Eppendorff tubes at concentrations of10^(8,) 10⁷ and 10⁶/ml in 100 μl medium. 100 μl of appropriate dilutionof ¹²⁵I-8H9 was added and allowed to react at 37° C. for 60 mins. Tubeswere subsequently centrifuged at 1400 rpm×10 mins. Radioactivity in 100μl of supernatant was counted using Minaxi gamma counter (PackardBioScience, Downer's Grove, Ill.) and compared with total counts in acontrol sample consisting of medium without cells. Percent binding wascalculated as (Experimental cpm/control cpm)×100%. The 8H9-negative cellline HTB119 was used as control.

Animal Studies

Biodistribution and Pharmacokinetics

All animal experiments were carried out under an IACUC approved protocoland institutional guidelines for the proper and humane use of animals inresearch were followed. Athymic nude mice (Ncr nu/nu) were purchasedfrom NCI, Frederick Md. They were xenografted subcutaneously with HTB82cell line (2×10⁶ cells/mouse) suspended in 100 ul of Matrigel(Beckton-Dickinson BioSciences, Bedford, Mass.) on the right flank.After 3-4 weeks, mice bearing tumors of 1 to 1.5 cm in longest dimensionwere selected. Mice were injected intravenously (retrorbital plexus)with 0.74 MBq or 4.44 MBq of ¹²⁵I-8H9, or with 4.44 MBq ¹²⁵I-2C9. Theywere anesthesized with ketamine (Fort Dodge Animal Health, Fort Dodge,Iowa) intraperitoneally and imaged at various time intervals with agamma camera (ADAC, Milpitas, Calif.) equipped with a high-resolutiongeneral-purpose collimator for ¹³¹I and thyroid X-ray grids for ¹²⁵I.Serial blood samples were collected at 5 min, 1, 2, 4, 8, 18, 24, 48,72, 120, 144 and 172 h to determine blood clearance of ¹²⁵I-8H9. Groupsof mice injected with ¹²¹I-8H9 were sacrificed at 24 h, 48 h, 120 h or172 h immediately after imaging. Mice injected with ¹²⁵I-2C9 were imagedeither at 120 h (and then sacrificed) or at 172 h. Samples of blood(cardiac sampling), heart, lung, liver, kidney, spleen, stomach,adrenal, small bowel, large bowel, spine, femur, muscle, skin, brain andtumor were weighed and radioactivity measured with a Minaxi-gammacounter. Results were expressed as percent injected dose per gram andbiodistnbution determined.

Toxicity

Athymic nude mice without xenografts were each injected with 4.44 MBq of¹³¹I-8H9. Groups of mice were euthanized at 15 and 30 days. Completeblood counts were carried out in each mouse via terminal bleed and liverfunction tests were performed on pooled sera. Complete necropsiesincluding gross and histological examinations were carried out toevaluate possible toxicity of ¹³¹I-8H9.

Evaluation of Anti-Tumor Activity

RMS xenografts were established as described above. Their maximalperpendicular axes were measured using calipers in control and tumorgroups. After 3 weeks, mice bearing tumors of approximately 0.7 cm³(tumor volume was calculated using the formula V=4πr³/3 where r=meanradius) were selected and injected with 18.5 MBq of ¹³¹I-8H9 or ¹³¹I-3F8(3F8 was used as a negative control antibody). Average serial tumorvolumes and body weights were monitored in the two groups and comparedover time. Mice were euthanized as per guidelines published in NIHPublication No. 85-23 (‘Principles of Laboratory Animal Care’). Data areexpressed as % increase or decrease in tumor volume when compared toinitial measurement on day 0 of treatment.

Results

Immunoreactivity

Protein bound ¹²⁵I and ¹³¹I as assessed by TCA precipitation averaged96±4.2% and 98±2.2 %, respectively for 8H9, and >95% for controlantibodies 2C9 and 3F8. TLC demonstrated free iodine peak of 1%, 99%being protein bound. Average maximum inmuunoreactivity as measured bysolid-phase RIA using the anti-8H9-idiotype 2E9 as antigen was 67±26%for 8H9 and 11% for 2C9. Maximum immunoreactivity measured by cellpellet binding assay was 83% for 8H9, maximum binding to the negativecontrol cell line HTB119 being 9%. 2C9 demonstrated maximum binding of6% on the HTB82 cell pellet.

Imaging

Animals tolerated intravenous injection without apparent ill effects.Tumor localization could be detected in animals imaged with ¹²⁵I-8H9 asearly as 4 hours after injection. At 24 h, tumor localization wasobvious along with some blood pool, liver and spleen uptake. At 48 h,blood pooling had significantly diminished and almost disappeared at 172h. In contrast, mice injected with the control IgG1 ¹²⁵I-2C9demonstrated no specific uptake in RMS xenografts (FIG. 1).

Blood Kinetics

Average blood clearance in groups of 5 mice with and without RMSxenografts injected with ¹²⁵I-8H9 is depicted in FIG. 2. Blood activityof ¹²⁵I-8H9 at 24 h was 14.3% and 17.3% injected dose per gm (% ID/g)respectively and dropped off to 3.3% and 5.3% ID/g, respectively at 172h. β half-life of ¹²⁵I-8H9 was 70.9 h.

Biodistribution

Table 2 lists the biodistribution of 4.44 MBq ¹²⁵I-8H9 in three groupsof mice with RMS xenografts studied at 24,48 and 172 h, respectively.Blood-pooling effect was observed at 24 h, which had diminished at 48 hand had almost completely subsided at 172 h after injection. There wasno significant activity in normal organs apart from blood at 172 h.Average tumor uptake was 11.5±3.9, 15.1±3.7, and 5.4±1.2 % injected doseper gm at 24, 48 and 172 h, respectively. Blood to tumor ratio was 1.24,0.71 and 0.59 at 24, 48 and 172 h respectively. Mean tumor/tissue ratios(FIG. 3) increased from 24 to 48 h and were optimal at 172 h (for lung,4, kidney 7, liver 8, spleen 11, femur 15, muscle 20, brain 47). In miceinjected with 0.74 MBq ¹²⁵I-8H9, there was a further increase intumor:tissue ratios particularly marked at 172 h post injection (forlung, 6, kidney 8, liver 12, spleen 14, femur 21, muscle 28, brain 56)(Table 3). Table 4 summarizes the biodistribution of ¹²⁵I-8H9 comparedto ¹²⁵I-2C9 at 120 h post injection. Average tumor uptake was 7.3±0.9%injected dose per gram for ¹²⁵I-8H9 as compared to 2.5±0.9% for¹²⁵I-2C9. Tumor to tissue ratios (FIG. 4) were <1 for almost all tissuesfor ¹²⁵I-2C9, as compared to 2.6-56.0 for ¹²⁵I-8H9.

Anti-Tumor Activity

Mice injected with 18.5 MBq ¹³¹I-8H9 showed a significant suppression intumor volume (FIG. 5). Average tumor volume had diminished to <50% ofinitial volume 21 days after injection. None of the tumors showed anyevidence of regrowth. In contrast, in the control group, mice injectedwith 18.5 MBq of ¹³¹I-3F8, an anti-GD2 MoAb that does not react withHTB82, there was progressive and rapid tumor growth.

Toxicity

No significant weight loss was noted in mice injected with 4.44 MBq of¹³¹I-8H9, 15 and 30 days post injection (data not shown). Complete bloodcount and liver function studies did not reveal any abnormalities (Table5). Complete necropsy evaluations did not reveal any gross orhistological lesions (data not shown). In the groups of mice treatedwith ¹³¹I labeled MoAbs, there was no significant weight loss 21 daysafter the initial dose for both the 3F8 and 8H9 groups (+11.7±8.8% forthe 3F8 group and −2±1.8% for the 8H9 group). The increase in weight inthe control group could be attributed to increasing tumor mass.

Discussion

Few tumor specific antigens that can be targeted by MoAbs have beendescribed for RMS. (Table 1) Myogenin, a myogenic regulatory proteinspecific for rhabdomyoblasts is nuclear in localization (16) andtherefore not amenable for targeting by MoAbs. Similarly, the MyoDfamily of oncofetal proteins is expressed in nuclei (17). Conversely,the cell membrane-expressed antigens, BW475 (18) and myosin (19), arealso expressed on normal neural and muscle tissue respectively. Thefetal form of the acetylcholine receptor, α2βγδ, a possible target forantibody-based immunotherapy, although not present on most normalmuscles tissue, is expressed in extraocular muscles, thymic myoid cellsand in denervated skeletal muscle. (20.) Blockade of the insulin-likegrowth factor I (IGFI) receptor, which has been implicated in anautocrine pathway in the growth of RMS by murine monoclonals has beendemonstrated to inhibit the growth of established RMS xenografts in nudemice (21). However, IGF receptors are ubiquitously expressed in normaltissues.

MoAb 8H9 recognizes a unique cell membrane antigen which is expressed ona wide range of pediatric and adult solid tumors (15). Furthermore, thisnovel antigen has restricted expression on normal tissues. In particularskeletal muscle and hematopoietic tissues are negative. Indeed 8H9 hasbeen utilized to purge Ewing's sarcoma from blood and bone marrow (26).In RMS, the 8H9 antigen is expressed on both alveolar and embryonalvariants. 96% (29/30) RMS studied expressed the 8H9 antigen. Expressionin most cases was strong and homogeneous. RMS cell lines including theHTB82 cell line have been shown to express this antigen on cellmembranes. It therefore has the potential to be utilized as a tumortarget in RMS.

RMS is a chemosensitive and radiosensitive tumor, yet in patients withmetastatic disease, MRD often leads to relapse and prevents cure.Immunotherapy using radiolabeled and unlabeled 8H9 may provide avaluable adjunct in the eradication of MRD. A similar approach has ledto successful cures being achieved in high-risk neuroblastoma (6). Inthis study we evaluated the in vivo targeting of RMS by radiolabeled8H9. We have demonstrated that radiolabeled 8H9 can be effectively usedin the radioimmunodetection and radioimmunotherapy of RMS xenografts inmice. Our results showed that ¹²⁵I or ¹³¹I labeled 8H9 retainedimmunoreactivity after radiolabeling. A relatively high specificactivity of >370 MBq/mg of ¹²⁵I was obtained without loss ofimmunoreactivity. Hence, 8H9 has the potential to be labeled withrelatively large doses of iodine radioisotopes for radioimmunotherapyapproaches.

Our imaging results show that 8H9 can specifically and selectively bindto human RMS xenografted in nude mice. Uptake in xenografts could bedetected as early as 4 h after injection. Excellent selectivity fortumor over normal tissue was demonstrated. There was no focal uptake inany normal organs including reticuloendothelial tissues. This is inkeeping with the specific distribution of the antigen recognized by 8H9as demonstrated by immunohistochemistry in human tissues and tumors(15). Specificity of 8H9 binding was demonstrated by comparing thebinding of ¹²⁵I-8H9 to that of ¹²⁵I-2C9. 2C9, an IgG1 MoAb specific forcytokeratin8, an antigen not expressed by the RMS cell line HTB82, wasused as a negative control. As expected, radiolabeled 2C9 remained inthe bloodstream and did not show any specific binding for RMS xenograftswith tumor: tissue ratios of 0.1-1. In comparison, radiation dose totumor relative to normal tissues for ¹²⁵I-8H9 ranged from 2.6 to 25.3fold. Specificity of ¹²⁵I-8H9 was also demonstrated in vitro by studyingthe binding of ¹²⁵I-8H9 to the 8H9 negative cell line HTB119 incomparison to the 8H9 positive line HTB82. Maximum binding of ¹²⁵I-8H9was 83% in comparison to 9% for HTBB119 indicating that ¹²⁵I-8H9 bindingwas antigen specific.

Biodistribution studies provided us with preclinical data inconsideration of a possible use for 8H9 in human trials. β half-life ofa single dose of 4.4 MBq of ²²⁵I-8H9 was 70.9 h. There was, in general,an excellent radiation dose differential between RMS and normal tissues.Optimum tumor to non-tumor ratios were reached at 172 h afterintravenous 8H9 injection. Blood:tumor ratios were relatively high at 24h indicative of blood pooling. Blood pooling diminished 48 h afterinjection and was further greatly reduced by 172 h. Probable uptake bycells of the reticuloendothelial system resulted in relatively highlevels for liver and spleen in the first24 h. There was increased uptakein the tumors at 48 h compared to 24 h suggesting further selectivetargeting of 8H9 between 24 to 48 h. Persistence of ¹²⁵I-8H9 in theblood during the first 48 h of administration implies that there is noappreciable neutralization of antibody by circulating 8H9 antigen. Whenlower doses of ¹²⁵I-8H9 for imaging (0.74MBq compared to 4.44 MBq),tumor: non-tumor ratios were improved, consistent with reduced bloodpooling (Table 4). The persistence of binding of ¹²⁵I-8H9 to tumorimplies that the 8H9 antigen is not immunomodulated off the cell afterantibody binding. Similar findings were demonstrated in vitro, where theantigen-antibody binding on cell surface as detected byimmunofluorescence persisted >60 h (15). This persistence should permita steady delivery of radiation to tumor cells by radiolabeled 8H9. Atdoses of 6.66 MBq/m², there were no clinical (body weights), chemical(CBC and LFTs) or gross or histologic organ toxicities at necropsies.

In an effort to develop systems to study antigen-antibody reactionspending the definitive identification of the glycosylated 58 kDa proteinantigen recognized by 8H9, we used anti-8H9-idiotypic MoAbs to serve assurrogate antigens. These have enabled us to study the binding ofradiolabeled (radioimmunoassay) and unlabeled (ELISA) 8H9 in vitro. Ourdata indicate that there was good correlation between the binding of¹²⁵I-8H9 to anti-8H9 anti-idiotypes and to native antigen on cellpellets.

The observed radioimmunotherapeutic effect of ¹³¹I-8H9 was remarkable,with >50% reduction in tumor volume of well-established RMS xenograftsbeing achieved with a dose of 18.5 MBq of ¹³¹I-8H9 without any adverseeffects. The antigen specific nature of this response was confirmed whenRMS xenografts treated with equivalent doses of nonspecific antibodydemonstrated unabated tumor growth. Radiolabeled 8H9 therefore, may havea possible clinical role in the therapy of RMS.

Given the broad reactivity of MoAb 8H9 with human solid tumors includingsarcomas, neuroblastoma and brain tumors, these studies provide theproof of principle for exploring antibody-based targeting strategiesdirected at this antigen.

REFERENCES

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1. A composition comprising an effective amount of monoclonal antibody8H9 or a derivative thereof and a suitable carrier.
 2. A pharmaceuticalcomposition comprising an effective amount of monoclonal antibody 8H9 ora derivative thereof and a pharmaceutically acceptable carrier.
 3. Thecomposition of claim 1 or 2, wherein the derivative is a scFv.
 4. Thecomposition of claim 3, wherein the antibody is an antibody-fusionconstruct
 5. The composition of claim 4, wherein the antibody is anscFv-Fc.
 6. An isolated antibody, wherein the Complementary DeterminingRegion is NYDIN, for CDR1, WIFPGDGSTQY for CDR2, QTTATVWFAY for CDR3 forthe heavy chain, and RASQSISDYLH for the CDR1, YASQSIS for CDR2,QNGHSFPLT for CDR3 for the light chain.
 7. The antibody of claim 6,wherein the other sequences are of human origin.
 8. The antibody ofclaim 1, which includes sequences as set forth in FIG.
 33. 9. Theantibody of claim 8, wherein the sequences are mutated.
 10. The antibodyof claim 9, which includes sequences as set forth in FIG.
 34. 11. Acomposition comprising the antibody of claim 6, 7, 8, 9, or
 10. 12. Anisolated nucleic acid molecule encoding the antibody of claim 6, 7, 8,9, or
 10. 13. The isolated nucleic acid of claim 6, wherein the sequenceis set forth in FIG.
 33. 14. A peptide or polypeptide comprising: NYDIN,WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, or QNGHSFPLT.
 15. Anisolated nucleic acid encoding the peptide of claim
 14. 16. A vectorcomprising the nucleic acid molecules of claim 12 or
 15. 17. A cellcomprising the vector of claim
 16. 18. An antibody other than themonoclonal antibody 8H9 comprising the complementary determining regionsof monoclonal antibody 8H9 or a derivative thereof, capable of bindingto the same antigen as the monoclonal antibody 8H9.
 19. A substancecapable of competitively inhibiting the binding of monoclonal antibody8H9.
 20. The substance of claim 19, which is an antibody.
 21. Anisolated single chain antibody of 8H9 or a derivative thereof.
 22. Anisolated scFv of monoclonal antibody 8H9 or a derivative thereof. 23.The isolated scFv of claim 21, wherein the scFv is directly orindirectly coupled to a cytotoxic agent.
 24. A cell comprising 8H9-scFv.25. The cell of claim 21, which is a red cell.
 26. A 8H9-scFv-genemodified cell.
 27. A liposome modified by 8H9-scFv.
 28. A method fordirectly killing, or delivering drug, DNA, RNA or derivatives thereof tocells bearing the antigen recognized by the monoclonal antibody 8H9 orto image cells or tumors bearing said antigen using the isolated scFv ofclaim 21 or 23 or cell or liposome of claim 23, 24, 25, or
 26. 29. Aprotein with about 58 kilodaltons in molecular weight, reactingspecifically with the monoclonal antibody 8H9.
 30. The protein of claim18, when glycosylated, the apparent molecular weight is about 90kilodaltons.
 31. An antibody produced by immunizing the protein of claim29 or specific portion thereof.
 32. A nucleic acid molecule encoding theprotein of claim
 29. 33. A nucleic acid molecule capable of specificallyhybridizing the molecule or claim
 32. 34. The nucleic acid moleculewherein it is synthetic DNA, genomic DNA, cDNA or RNA.
 35. A vectorcomprising the nucleic acid molecule of claim 32 or a portion thereof.36. A cell comprising the nucleic acid molecule of claim
 32. 37. Amethod for producing the protein which binds to the monoclonal antibody8H9 comprising cloning the nucleic acid molecule of claim 32 in anappropriate vector, expressing said protein in appropriate cells andrecovery of said expressed protein.
 38. A method for production ofantibody using the protein produced by the method of claim
 37. 39. Theantibody produced by the method of claim
 38. 40. The antibody of claim39, wherein the antibody is a monoclonal.
 41. A method of inhibiting thegrowth of tumor cells comprising contacting said tumor cells with anappropriate amount of monoclonal antibody 8H9 or a derivative thereof,or the antibody of claim 39 or a derivative thereof.
 42. A method ofinhibiting the growth of tumor cells in a subject comprisingadministering to the subject an appropriate amount of monoclonalantibody 8H9 or a derivative thereof, or the antibody of claim 39 or aderivative thereof.
 43. A method for imaging a tumor in a subjectcomprising administering to the subject a labeled monoclonal antibody8H9 or a labeled derivatives, or a labeled antibody of claim 39 or alabeled derivative.
 44. A method of reducing tumor cells in a subjectcomprising administering to the subject monoclonal antibody 8H9 or aderivative thereof, or a monoclonal antibody of claim 40 or a derivativethereof wherein the antibody or derivative is coupled to a cytotoxicagent to the subject.
 45. The method of claim 44, wherein the couplingto a cytotoxic agent is indirect.
 46. The method of claim 44, whereinthe coupling is to link the antibody or derivative with a first proteincapable of binding to a second protein and the second protein iscovalently coupled to a cytotoxic agent.
 47. The method of claim 44,wherein the cytotoxic agent is a radioisotope.
 48. A method to evaluatethe tumor bearing potential of a subject comprising measuring theexpression the 8H9 antigen in the subject, wherein the increasedexpression of said antigen indicates higher tumor bearing potential ofthe subject.
 49. A transgenic animal comprising an exogenous geneencoding the 8H9 antigen.
 50. The transgenic animal wherein the geneencoding the 8H9 mouse analogous antigen has been knocked out.
 51. Thetransgenic mouse of claim
 49. 52. A method to screening new anti-tumorcompound comprising contacting the transgenic animal of claim 49 withthe tested compound and measuring the level of expression of the 8H9antigen in said transgenic animal, a decrease in the level of expressionindicating that the compound can inhibit the expression of the 8H9antigen and is a anti-tumor candidate.